Oxford Nanopore R10.4 long-read sequencing enables the generation of near-finished bacterial genomes from pure cultures and metagenomes without short-read or reference polishing.

Long-read Oxford Nanopore sequencing has democratized microbial genome sequencing and enables the recovery of highly contiguous microbial genomes from isolates or metagenomes. However, to obtain near-finished genomes it has been necessary to include short-read polishing to correct insertions and deletions derived from homopolymer regions. Here, we show that Oxford Nanopore R10.4 can be used to generate near-finished microbial genomes from isolates or metagenomes without short-read or reference polishing.

Mass-immigration determines the assembly of activated sludge microbial communities.

The assembly of bacterial communities in wastewater treatment plants (WWTPs) is affected by immigration via wastewater streams, but the impact and extent of bacterial immigrants are still unknown. Here, we quantify the effect of immigration at the species level in 11 Danish full-scale activated sludge (AS) plants. All plants have different source communities but have very similar process design, defining the same overall environmental growth conditions. The AS community composition in each plant was strongly reflected by the corresponding influent wastewater (IWW) microbial composition. Most species in AS across the plants were detected and quantified in the corresponding IWW, allowing us to identify their fate in the AS: growing, disappearing, or surviving. Most of the abundant species in IWW disappeared in AS, so their presence in the AS biomass was only due to continuous mass-immigration. In AS, most of the abundant growing species were present in the IWW at very low abundances. We predicted the AS species abundances from their abundance in IWW by using a partial least square regression model. Some species in AS were predicted by their own abundance in IWW, while others by multiple species abundances. Detailed analyses of functional guilds revealed different prediction patterns for different species. We show, in contrast to the present understanding, that the AS microbial communities were strongly controlled by the IWW source community and could be quantitatively predicted by taking into account immigration. This highlights a need to revise the way we understand, design, and manage the microbial communities in WWTPs.

Regression on imperfect class labels derived by unsupervised clustering.

Outcome regressed on class labels identified by unsupervised clustering is custom in many applications. However, it is common to ignore the misclassification of class labels caused by the learning algorithm, which potentially leads to serious bias of the estimated effect parameters. Due to their generality we suggest to address the problem by use of regression calibration or the misclassification simulation and extrapolation method. Performance is illustrated by simulated data from Gaussian mixture models, documenting a reduced bias and improved coverage of confidence intervals when adjusting for misclassification with either method. Finally, we apply our method to data from a previous study, which regressed overall survival on class labels derived from unsupervised clustering of gene expression data from bone marrow samples of multiple myeloma patients.

Infection, recovery and re-infection of farmed mink with SARS-CoV-2.

Mink, on a farm with about 15,000 animals, became infected with SARS-CoV-2. Over 75% of tested animals were positive for SARS-CoV-2 RNA in throat swabs and 100% of tested animals were seropositive. The virus responsible had a deletion of nucleotides encoding residues H69 and V70 within the spike protein gene as well as the A22920T mutation, resulting in the Y453F substitution within this protein, seen previously in mink. The infected mink recovered and after free-testing of 300 mink (a level giving 93% confidence of detecting a 1% prevalence), the animals remained seropositive. During further follow-up studies, after a period of more than 2 months without any virus detection, over 75% of tested animals again scored positive for SARS-CoV-2 RNA. Whole genome sequencing showed that the viruses circulating during this re-infection were most closely related to those identified in the first outbreak on this farm but additional sequence changes had occurred. Animals had much higher levels of anti-SARS-CoV-2 antibodies in serum samples after the second round of infection than at free-testing or during recovery from initial infection, consistent with a boosted immune response. Thus, it was concluded that following recovery from an initial infection, seropositive mink were readily re-infected by SARS-CoV-2.

Clinical results and microbiota changes after faecal microbiota transplantation for chronic pouchitis: a pilot study.

Objectives: Research evidence suggests that chronic pouchitis is associated with intestinal dysbiosis. Faecal microbiota transplantation (FMT) has been proposed as a possible treatment. We performed a 6-month prospective, open-label, single-centre cohort pilot-study (NCT03538366) to investigate if FMT could improve clinical outcome and alter gut microbiota in patients with chronic pouchitis.Materials and methods: Nine adult patients with chronic pouchitis were included and allocated to 14 days FMT by enemas from five faecal donors, with a 6-month follow-up. Pouchitis severity was assessed using pouchitis disease activity index (PDAI) before and after FMT. Changes in gut microbiota, and engraftment of donor's microbiota were assessed in faecal samples.Results: All patients were treated with FMT for 14 continuous days. Overall, four of nine patients receiving FMT were in clinical remission at 30-day follow-up, and three patients remained in remission until 6-month follow-up. Clinical symptoms of pouchitis improved significantly between inclusion and 14-day follow-up (p = .02), but there was no improvement in PDAI between inclusion (mean 8.6) and 30-day follow-up (mean 5.2). Treatment with FMT caused a substantial shift in microbiota and increased microbial diversity in six patients, resembling that of the donors, with a high engraftment of specific donor microbiota.Conclusions: Symptomatic benefit in FMT treatment was found for four of nine patients with chronic pouchitis with increased microbial diversity and high engraftment of donor's microbiota. A larger, randomised controlled study is required to fully evaluate the potential role of FMT in treating chronic pouchitis.

Automated swimming activity monitor for examining temporal patterns of toxicant effects on individual Daphnia magna.

Aquatic pollutants are often biologically active at low concentrations and impact on biota in combination with other abiotic stressors. Traditional toxicity tests may not detect these effects, and there is a need for sensitive high-throughput methods for detecting sublethal effects. We have evaluated an automated infra-red (IR) light-based monitor for recording the swimming activity of Daphnia magna to establish temporal patterns of toxicant effects on an individual level. Activity was recorded for 48 h and the sensitivity of the monitor was evaluated by exposing D. magna to the reference chemicals K2 Cr2 O7 at 15, 20 and 25 °C and 2,4-dichlorophenol at 20 °C. Significant effects (P < 0.001) of toxicant concentrations, exposure time and incubation temperatures were observed. At 15 °C, the swimming activity remained unchanged for 48 h at sublethal concentrations of K2 Cr2 O7 whereas activity at 20 and 25 °C was more biphasic with decreases in activity occurring after 12-18 h. A similar biphasic pattern was observed after 2,4-dichlorophenol exposure at 20 °C. EC50 values for 2,4-dichlorophenol and K2 Cr2 O7 determined from automated recording of swimming activity showed increasing toxicity with time corresponding to decreases in EC50 of 0.03-0.07 mg l(-1) h(-1) . EC50 values determined after 48 h were comparable or lower than EC50 values based on visual inspection according to ISO 6341. The results demonstrated that the swimming activity monitor is capable of detecting sublethal behavioural effects that are toxicant and temperature dependent. The method allows EC values to be established at different time points and can serve as a high-throughput screening tool in toxicity testing. Copyright © 2015 John Wiley & Sons, Ltd.

Full-scale activated sludge transplantation reveals a highly resilient community structure.

Well-functioning and stable microbial communities are critical for the operation of activated sludge (AS) wastewater treatment plants (WWTPs). Bioaugmentation represents a potentially useful approach to recover deteriorated systems or to support specific AS processes, but its application in full-scale WWTPs is generally problematic. We conducted a massive transplantation (in one day) exchanging AS from a donor to a recipient full-scale WWTP with similar process type (biological removal of nitrogen and phosphorus) and performance, but with differences in microbial community structure. The treatment performance in the recipient plant was not compromised and the effluent quality remained stable. The AS community structure of the recipient plant was initially very similar to the donor AS, but it almost completely restored the pre-transplantation structure approximately 40 days after transplantation, corresponding to 3 times the solid retention time. Most of the unique species of donor AS added to recipient AS disappeared quickly, although some disappeared more slowly the following months, indicating some survival and potentially a time limited function in the recipient plant. Moreover, the addition in higher abundance of most species already present in the recipient AS (e.g., the polyphosphate accumulating organisms) or the reduction of the abundance of unwanted bacteria (e.g., filamentous bacteria) in the recipient plant was not successful. Moreover, we observed similar abundance patterns after transplantation for species belonging to different functional guilds, so we did not observe an increase of the functional redundancy. Investigations of the microbial community structure in influent wastewater revealed that for some species the abundance trends in the recipient plant were closely correlated to their abundance in the influent. We showed that a very resilient microbial community was responsible for the outcome of the transplantation of AS at full-scale WWTP, potentially as a consequence of mass-immigration from influent wastewater. The overall results imply that massive transplantation of AS across different WWTPs is not a promising strategy to permanently solve operational problems. However, by choosing a compatible AS donor, short term mitigation of serious operational problems may be possible.

Faecal microbiota transplantation from patients with depression or healthy individuals into rats modulates mood-related behaviour.

Differences in gut microbiota composition have been observed in patients with major depressive disorder (MDD) compared to healthy individuals. Here, we investigated if faecal microbiota transplantation (FMT) from patients with MDD into rats could induce a depressive-like phenotype. We performed FMT from patients with MDD (FMT-MDD) and healthy individuals (FMT-Healthy) into male Flinders Sensitive Line (FSL) and Flinders Resistant Line (FRL) rats and assessed depressive-like behaviour. No behavioural differences were observed in the FSL rats. In FRL rats, the FMT-Healthy group displayed significantly less depressive-like behaviour than the FMT-MDD group. However, there was no difference in behaviour between FMT-MDD FRL rats and negative controls, indicating that FMT-Healthy FRL rats received beneficial bacteria. We additionally found different taxa between the FMT-MDD and the FMT-Healthy FRL rats, which could be traced to the donors. Four taxa, three belonging to the family Ruminococcaceae and the genus Lachnospira, were significantly elevated in relative abundance in FMT-MDD rats, while the genus Coprococcus was depleted. In this study, the FMT-MDD group was different from the FMT-Healthy group based on behaviour and intestinal taxa.

Increased transmissibility of SARS-CoV-2 lineage B.1.1.7 by age and viral load.

New lineages of SARS-CoV-2 are of potential concern due to higher transmissibility, risk of severe outcomes, and/or escape from neutralizing antibodies. Lineage B.1.1.7 (the Alpha variant) became dominant in early 2021, but the association between transmissibility and risk factors, such as age of primary case and viral load remains poorly understood. Here, we used comprehensive administrative data from Denmark, comprising the full population (January 11 to February 7, 2021), to estimate household transmissibility. This study included 5,241 households with primary cases; 808 were infected with lineage B.1.1.7 and 4,433 with other lineages. Here, we report an attack rate of 38% in households with a primary case infected with B.1.1.7 and 27% in households with other lineages. Primary cases infected with B.1.1.7 had an increased transmissibility of 1.5-1.7 times that of primary cases infected with other lineages. The increased transmissibility of B.1.1.7 was multiplicative across age and viral load.

Risk of hospitalisation associated with infection with SARS-CoV-2 lineage B.1.1.7 in Denmark: an observational cohort study.

BACKGROUND: The more infectious SARS-CoV-2 lineage B.1.1.7 rapidly spread in Europe after December, 2020, and a concern that B.1.1.7 could cause more severe disease has been raised. Taking advantage of Denmark's high RT-PCR testing and whole genome sequencing capacities, we used national health register data to assess the risk of COVID-19 hospitalisation in individuals infected with B.1.1.7 compared with those with other SARS-CoV-2 lineages. METHODS: We did an observational cohort study of all SARS-CoV-2-positive cases confirmed by RT-PCR in Denmark, sampled between Jan 1 and March 24, 2021, with 14 days of follow-up for COVID-19 hospitalisation. Cases were identified in the national COVID-19 surveillance system database, which includes data from the Danish Microbiology Database (RT-PCR test results), the Danish COVID-19 Genome Consortium, the National Patient Registry, the Civil Registration System, as well as other nationwide registers. Among all cases, COVID-19 hospitalisation was defined as first admission lasting longer than 12 h within 14 days of a sample with a positive RT-PCR result. The study population and main analysis were restricted to the proportion of cases with viral genome data. We calculated the risk ratio (RR) of admission according to infection with B.1.1.7 versus other co-existing lineages with a Poisson regression model with robust SEs, adjusted a priori for sex, age, calendar time, region, and comorbidities. The contribution of each covariate to confounding of the crude RR was evaluated afterwards by a stepwise forward inclusion. FINDINGS: Between Jan 1 and March 24, 2021, 50 958 individuals with a positive SARS-CoV-2 test and at least 14 days of follow-up for hospitalisation were identified; 30 572 (60·0%) had genome data, of whom 10 544 (34·5%) were infected with B.1.1.7. 1944 (6·4%) individuals had a COVID-19 hospitalisation and of these, 571 (29·4%) had a B.1.1.7 infection and 1373 (70·6%) had an infection with other SARS-CoV-2 lineages. Although the overall number of hospitalisations decreased during the study period, the proportion of individuals infected with B.1.1.7 increased from 3·5% to 92·1% per week. B.1.1.7 was associated with a crude RR of hospital admission of 0·79 (95% CI 0·72-0·87; p<0·0001) and an adjusted RR of 1·42 (95% CI 1·25-1·60; p<0·0001). The adjusted RR was increased in all strata of age and calendar period-the two covariates with the largest contribution to confounding of the crude RR. INTERPRETATION: Infection with SARS-CoV-2 lineage B.1.1.7 was associated with an increased risk of hospitalisation compared with that of other lineages in an analysis adjusted for covariates. The overall effect on hospitalisations in Denmark was lessened due to a strict lockdown, but our findings could support hospital preparedness and modelling of the projected impact of the epidemic in countries with uncontrolled spread of B.1.1.7. FUNDING: None.

The Microbiota Profile in Inflamed and Non-Inflamed Ileal Pouch-Anal Anastomosis.

The objective was to determine the bacterial composition in inflamed and non-inflamed pouches for comparison to the microbiota of healthy individuals. Pouch patients and healthy individuals were included between November 2017 and June 2019 at the Department of Gastrointestinal Surgery, Aalborg University Hospital, Denmark. A faecal sample was collected from all participants for microbiota analysis using 16S rRNA amplicon sequencing. Overall, 38 participants were included in the study. Eleven patients with a normally functioning pouch, 9 patients with chronic pouchitis, 6 patients with familial adenomatous polyposis, and 12 healthy individuals. Patients with chronic pouchitis had overall lower microbial diversity and richness compared to patients with a normal pouch function (p < 0.001 and p = 0.009) and healthy individuals (p < 0.001 and p < 0.001). No significant difference was found between patients with familial adenomatous polyposis and chronic pouchitis (microbial diversity p = 0.39 and richness p = 0.78). Several taxa from the family Enterobacteriaceae, especially genus Escherichia, were associated primarily with patients with chronic pouchitis, while taxa from the genus Bacteroides primarily were associated with healthy individuals and patients with a normally functioning pouch. Finally, a microbial composition gradient could be established from healthy individuals through patients with normal pouch function and familial adenomatous polyposis to patients with chronic pouchitis.

Housefly (Musca domestica L.) associated microbiota across different life stages.

The housefly (Musca domestica L.) lives in close association with its microbiota and its symbionts are suggested to have pivotal roles in processes such as metabolism and immune response, but it is unclear how the profound physiological changes during ontogeny affect the housefly's associated microbiota and their metabolic capabilities. The present study applies 16S rRNA gene amplicon sequencing to investigate the development of the host-associated microbiota during ontogeny. The potential for microbiota transfer between developmental stages, and the metabolic potential of these microbiota were evaluated. Representatives of Firmicutes were observed as early colonisers during the larval stages, followed by colonisation by organisms affiliating with Proteobacteria and Bacteroidetes as the flies matured into adults. Microbiota observed across all the developmental stages included Lactococcus, Lactobacillus and Enterococcus, while Weissella and Chishuiella were associated with newly hatched larvae and adults, respectively. Predictive metabolic profiling of the identified microorganisms further suggested that the microbiota and their functional profile mature alongside their host and putative host-microbe relationships are established at different stages of development. The predicted metabolic capability of the microbiota developed from primarily simple processes including carbohydrate and nucleotide metabolisms, to more complex metabolic pathways including amino acid metabolisms and processes related to signal transduction.

Novel syntrophic bacteria in full-scale anaerobic digesters revealed by genome-centric metatranscriptomics.

Short-chain fatty acid (SCFA) degradation is an important process in methanogenic ecosystems, and is usually catalyzed by SCFA-oxidizing bacteria in syntrophy with methanogens. Current knowledge of this functional guild is mainly based on isolates or enrichment cultures, but these may not reflect the true diversity and in situ activities of the syntrophs predominating in full-scale systems. Here we obtained 182 medium to high quality metagenome-assembled genomes (MAGs) from the microbiome of two full-scale anaerobic digesters. The transcriptomic response of individual MAG was studied after stimulation with low concentrations of acetate, propionate, or butyrate, separately. The most pronounced response to butyrate was observed for two MAGs of the recently described genus Candidatus Phosphitivorax (phylum Desulfobacterota), expressing a butyrate beta-oxidation pathway. For propionate, the largest response was observed for an MAG of a novel genus in the family Pelotomaculaceae, transcribing a methylmalonyl-CoA pathway. All three species were common in anaerobic digesters at Danish wastewater treatment plants as shown by amplicon analysis, and this is the first time their syntrophic features involved in SCFA oxidation were revealed with transcriptomic evidence. Further, they also possessed unique genomic features undescribed in well-characterized syntrophs, including the metabolic pathways for phosphite oxidation, nitrite and sulfate reduction.

The Signal and the Noise: Characteristics of Antisense RNA in Complex Microbial Communities.

High-throughput sequencing has allowed unprecedented insight into the composition and function of complex microbial communities. With metatranscriptomics, it is possible to interrogate the transcriptomes of multiple organisms simultaneously to get an overview of the gene expression of the entire community. Studies have successfully used metatranscriptomics to identify and describe relationships between gene expression levels and community characteristics. However, metatranscriptomic data sets contain a rich suite of additional information that is just beginning to be explored. Here, we focus on antisense expression in metatranscriptomics, discuss the different computational strategies for handling it, and highlight the strengths but also potentially detrimental effects on downstream analysis and interpretation. We also analyzed the antisense transcriptomes of multiple genomes and metagenome-assembled genomes (MAGs) from five different data sets and found high variability in the levels of antisense transcription for individual species, which were consistent across samples. Importantly, we challenged the conceptual framework that antisense transcription is primarily the product of transcriptional noise and found mixed support, suggesting that the total observed antisense RNA in complex communities arises from the combined effect of unknown biological and technical factors. Antisense transcription can be highly informative, including technical details about data quality and novel insight into the biology of complex microbial communities.IMPORTANCE This study systematically evaluated the global patterns of microbial antisense expression across various environments and provides a bird's-eye view of general patterns observed across data sets, which can provide guidelines in our understanding of antisense expression as well as interpretation of metatranscriptomic data in general. This analysis highlights that in some environments, antisense expression from microbial communities can dominate over regular gene expression. We explored some potential drivers of antisense transcription, but more importantly, this study serves as a starting point, highlighting topics for future research and providing guidelines to include antisense expression in generic bioinformatic pipelines for metatranscriptomic data.

A B-cell-associated gene signature classification of diffuse large B-cell lymphoma by NanoString technology.

Gene expression profiling (GEP) by microarrays of diffuse large B-cell lymphoma (DLBCL) has enabled the categorization of DLBCL into activated B-cell-like and germinal center B-cell-like subclasses. However, as this does not fully embrace the great diversity of B-cell subtypes, we recently developed a gene expression assay for B-cell-associated gene signature (BAGS) classification. To facilitate quick and easy-to-use BAGS profiling, we developed in this study the NanoString-based BAGS2Clinic assay. Microarray data from 4 different cohorts (n = 970) were used to select genes and train the assay. The locked assay was validated in an independent cohort of 88 sample biopsies. The assay showed good correspondence with the original BAGS classifier, with an overall accuracy of 84% (95% confidence interval, 72% to 93%) and a subtype-specific accuracy ranging between 80% and 99%. BAGS classification has the potential to provide valuable insight into tumor biology as well as differences in resistance to immuno- and chemotherapy that can lead to novel treatment strategies for DLBCL patients. BAGS2Clinic can facilitate this and the implementation of BAGS classification as a routine clinical tool to improve prognosis and treatment guidance for DLBCL patients.

The number of extranodal sites assessed by PET/CT scan is a powerful predictor of CNS relapse for patients with diffuse large B-cell lymphoma: An international multicenter study of 1532 patients treated with chemoimmunotherapy.

PURPOSE: Development of secondary central nervous system involvement (SCNS) in patients with diffuse large B-cell lymphoma is associated with poor outcomes. The CNS International Prognostic Index (CNS-IPI) has been proposed for identifying patients at greatest risk, but the optimal model is unknown. METHODS: We retrospectively analysed patients with diffuse large B-cell lymphoma diagnosed between 2001 and 2013, staged with PET/CT and treated with R-CHOP(-like) regimens. Baseline clinicopathologic characteristics, treatments, and outcome data were collected from clinical databases and medical files. We evaluated the association between candidate prognostic factors and modelled different risk models for predicting SCNS. RESULTS: Of 1532 patients, 62 (4%) subsequently developed SCNS. By multivariate analysis, disease stage III/IV, elevated serum LDH, kidney/adrenal and uterine/testicular involvement were independently associated with SCNS. There was a strong correlation between absolute number of extranodal sites and risk of SCNS; the 144 patients (9%) with >2 extranodal sites had a 3-year cumulative incidence of SCNS of 15.2% (95% confidence interval [CI] 9.2-21.2%) compared with 2.6% (95% CI 1.7-3.5) among those with ≤2 sites (P < 0.001). The 3-year cumulative risks of SCNS for CNS-IPI defined risk groups were 11.2%, 3.1% and 0.4% for high-, intermediate- and low-risk patients, respectively. All risk models analysed had high negative predictive values, but only modest positive predictive values. CONCLUSIONS: Patients with >2 extranodal sites or high-risk disease according to the CNS-IPI should be considered for baseline CNS staging. Clinical risk prediction models suffer from limited positive predictive ability, highlighting the need for more sensitive biomarkers to identify patients at highest risk of this devastating complication.