Acquired aplastic anemia following SARS-CoV-2 vaccination.

COVID-19 is a potential life-threatening viral disease caused by SARS-CoV-2 and was declared a pandemic by the WHO in March 2020. mRNA-based SARS-CoV-2 vaccines are routinely recommended in immune-compromised patients, including patients with AA, as these patients are at increased risk of contracting COVID-19 and developing a more severe course of disease. Between March 2021 and November 2021 relapse of AA occurred in four (age [median]: 53 years, range 30-84 years) out of 135 patients currently registered at our department and two de novo cases of AA in temporal context to vaccination against SARS-CoV-2, were documented. Median time after first COVID-19 vaccination and relapse of AA was 77 days. All relapsed patients were vaccinated with the mRNA-based vaccine Comirnaty®. Relapse in two out of the four patients was refractory to CsA/eltrombopag, favoring IST with hATG/CsA or BMT, respectively. Our observations should prompt clinicians to take vaccine-induced relapse of AA or de novo AA after SARS-CoV-2 vaccination into account. Furthermore, careful clinical monitoring and vigilance for signs or symptoms that may indicate relapse of AA (e.g., bleeding complications) are indicated.

Newly identified proviruses in Thermotogota suggest that viruses are the vehicles on the highways of interphylum gene sharing.

Phylogenomic analyses of bacteria from the phylum Thermotogota have shown extensive lateral gene transfer with distantly related organisms, particularly with Firmicutes. One likely mechanism of such DNA transfer is viruses. However, to date, only three temperate viruses have been characterized in this phylum, all infecting bacteria from the Marinitoga genus. Here we report 17 proviruses integrated into genomes of bacteria belonging to eight Thermotogota genera and induce viral particle production from one of the proviruses. All except an incomplete provirus from Mesotoga fall into two groups based on sequence similarity, gene synteny and taxonomic classification. Proviruses of Group 1 are found in the genera Geotoga, Kosmotoga, Marinitoga, Thermosipho and Mesoaciditoga and are similar to the previously characterized Marinitoga viruses, while proviruses from Group 2 are distantly related to the Group 1 proviruses, have different genome organization and are found in Petrotoga and Defluviitoga. Genes carried by both groups are closely related to Firmicutes and Firmicutes (pro)viruses in phylogenetic analyses. Moreover, one of the groups show evidence of recent gene exchange and may be capable of infecting cells from both phyla. We hypothesize that viruses are responsible for a large portion of the observed gene flow between Firmicutes and Thermotogota.

Metagenomic Shotgun Analyses Reveal Complex Patterns of Intra- and Interspecific Variation in the Intestinal Microbiomes of Codfishes.

The relative importance of host-specific selection or environmental factors in determining the composition of the intestinal microbiome in wild vertebrates remains poorly understood. Here, we used metagenomic shotgun sequencing of individual specimens to compare the levels of intra- and interspecific variation of intestinal microbiome communities in two ecotypes (NEAC and NCC) of Atlantic cod (Gadus morhua) that have distinct behavior and habitats and three Gadidae species that occupy a range of ecological niches. Interestingly, we found significantly diverged microbiomes among the two Atlantic cod ecotypes. Interspecific patterns of variation are more variable, with significantly diverged communities for most species' comparisons, apart from the comparison between coastal cod (NCC) and Norway pout (Trisopterus esmarkii), whose community compositions are not significantly diverged. The absence of consistent species-specific microbiomes suggests that external environmental factors, such as temperature, diet, or a combination thereof, comprise major drivers of the intestinal community composition of codfishes.IMPORTANCE The composition of the intestinal microbial community associated with teleost fish is influenced by a diversity of factors, ranging from internal factors (such as host-specific selection) to external factors (such as niche occupation). These factors are often difficult to separate, as differences in niche occupation (e.g., diet, temperature, or salinity) may correlate with distinct evolutionary trajectories. Here, we investigate four gadoid species with contrasting levels of evolutionary separation and niche occupation. Using metagenomic shotgun sequencing, we observed distinct microbiomes among two Atlantic cod (Gadus morhua) ecotypes (NEAC and NCC) with distinct behavior and habitats. In contrast, interspecific patterns of variation were more variable. For instance, we did not observe interspecific differentiation between the microbiomes of coastal cod (NCC) and Norway pout (Trisopterus esmarkii), whose lineages underwent evolutionary separation over 20 million years ago. The observed pattern of microbiome variation in these gadoid species is therefore most parsimoniously explained by differences in niche occupation.

Defective migration and dysmorphology of neutrophil granulocytes in atypical chronic myeloid leukemia treated with ruxolitinib.

BACKGROUND: The identification of pathologically altered neutrophil granulocyte migration patterns bears strong potential for surveillance and prognostic scoring of diseases. We recently identified a strong correlation between impaired neutrophil motility and the disease stage of myelodysplastic syndrome (MDS). Here, we apply this assay to study quantitively increased neutrophils of a patient suffering from a rare leukemia subtype, atypical chronic myeloid leukemia (aCML). METHODS: A 69-year-old male was analyzed in this study. Besides routine analyses, we purified the patient's neutrophils from peripheral whole blood and studied their migration behavior using time-lapse video microscopy in a standardized assay. These live cell migration analyses also allowed for the quantification of cell morphology. Furthermore, the cells were stained for the markers CD15, CD16, fMLPR, CXCR1 and CXCR2. RESULTS: Despite cytoreductive therapy with hydroxyurea, the patient's WBC and ANC were poorly controlled and severe dysgranulopoiesis with hypogranularity was observed. Neutrophils displayed strongly impaired migration when compared to healthy controls and migrating cells exhibited a more flattened-out morphology than control neutrophils. Because of a detected CSF3R (p.T618I) mutation and constitutional symptoms treatment with ruxolitinib was initiated. Within 1 week of ruxolitinib treatment, the cell shape normalized and remained indistinguishable from healthy control neutrophils. However, neutrophil migration did not improve over the course of ruxolitinib therapy but was strikingly altered shortly before a sinusitis with fever and bleeding from a gastric ulcer. Molecular work-up revealed that under ruxolitinib treatment, the CSF3R clone was depleted, yet the expansion of a NRAS mutated subclone was promoted. CONCLUSION: These results demonstrate the usefulness of neutrophil migration analyses to uncover corresponding alterations of neutrophil migration in rare myeloid neoplasms. Furthermore, in addition to monitoring migration the determination of morphological features of live neutrophils might represent a useful tool to monitor the effectiveness of therapeutic approaches.

Switching on the light: using metagenomic shotgun sequencing to characterize the intestinal microbiome of Atlantic cod.

Atlantic cod (Gadus morhua) is an ecologically important species with a wide-spread distribution in the North Atlantic Ocean, yet little is known about the diversity of its intestinal microbiome in its natural habitat. No geographical differentiation in this microbiome was observed based on 16S rRNA amplicon analyses, yet such finding may result from an inherent lack of power of this method to resolve fine-scaled biological complexity. Here, we use metagenomic shotgun sequencing to investigate the intestinal microbiome of 19 adult Atlantic cod individuals from two coastal populations in Norway-located 470 km apart. Resolving the species community to unprecedented resolution, we identify two abundant species, Photobacterium iliopiscarium and Photobacterium kishitanii, which comprise over 50% of the classified reads. Interestingly, the intestinal P. kishitanii strains have functionally intact lux genes, and its high abundance suggests that fish intestines form an important part of its ecological niche. These observations support a hypothesis that bioluminescence plays an ecological role in the marine food web. Despite our improved taxonomical resolution, we identify no geographical differences in bacterial community structure, indicating that the intestinal microbiome of these coastal cod is colonized by a limited number of closely related bacterial species with a broad geographical distribution.

Genomic analysis of the mesophilic Thermotogae genus Mesotoga reveals phylogeographic structure and genomic determinants of its distinct metabolism.

The genus Mesotoga, the only described mesophilic Thermotogae lineage, is common in mesothermic anaerobic hydrocarbon-rich environments. Besides mesophily, Mesotoga displays lineage-specific phenotypes, such as no or little H2 production and dependence on sulfur-compound reduction, which may influence its ecological role. We used comparative genomics of 18 Mesotoga strains (pairwise 16S rRNA identity >99%) and a transcriptome of M. prima to investigate how life at moderate temperatures affects phylogeography and to interrogate the genomic features of its lineage-specific metabolism. We propose that Mesotoga accomplish H2 oxidation and thiosulfate reduction using a sulfide dehydrogenase and a hydrogenase-complex and that a pyruvate:ferredoxin oxidoreductase acquired from Clostridia is responsible for oxidizing acetate. Phylogenetic analysis revealed three distinct Mesotoga lineages (89.6%-99.9% average nucleotide identity [ANI] within lineages, 79.3%-87.6% ANI between lineages) having different geographic distribution patterns and high levels of intra-lineage recombination but little geneflow between lineages. Including data from metagenomes, phylogeographic patterns suggest that geographical separation historically has been more important for Mesotoga than hyperthermophilic Thermotoga and we hypothesize that distribution of Mesotoga is constrained by their anaerobic lifestyle. Our data also suggest that recent anthropogenic activities and environments (e.g., wastewater treatment, oil exploration) have expanded Mesotoga habitats and dispersal capabilities.

Acute porphyrias: a German monocentric study of the biochemical, molecular genetic, and clinical data of 62 families.

In Germany, analyses of clinical and laboratory features of patients with acute porphyrias are only available for hereditary coproporphyria (HCP) but not with other acute porphyrias, acute intermittent porphyria (AIP) and variegate porphyria (VP). The aim of the study was to analyze a large cohort of patients with particular focus upon quality of life aspects. Sixty-two individuals from separate families with acute porphyrias (57 AIP, 5 VP) were included into an observational study collecting biochemical, genetic, and clinical data. A questionnaire was designed to complete anamnestic information and to assess the influence on quality of life. Most frequent signs and symptoms or laboratory abnormalities were abdominal colicky pain, red coloration of urine, and hyponatremia. Depression or anxiety was reported by 61% or 52% individuals, respectively. Fatigue was mentioned as the most quality of life-limiting symptom. In 59/61 patients, mutations could be identified. 44% (20/45) had to be admitted to an intensive care unit. Heme arginate was used in 64% (29/45) of patients for treatment of acute attacks at least once and in 33% for long-term treatment with high frequency of administration. Serum creatinine values increased in 47% (7/17) of the patients with recurrent attacks. Our analysis confirms a substantial influence of the diseases on the quality of life on patients. Percentages of urine discoloration and intensive care unit admissions were much higher than in other reports. Long-term treatment with heme arginate requires careful monitoring of iron status and renal values.

Gastrointestinal microbial community changes in Atlantic cod (Gadus morhua) exposed to crude oil.

BACKGROUND: The expansion of offshore oil exploration increases the risk of marine species being exposed to oil pollution in currently pristine areas. The adverse effects of oil exposure through toxic properties of polycyclic aromatic hydrocarbons (PAHs) have been well studied in Atlantic cod (Gadus morhua). Nevertheless, the fate of conjugated metabolites in the intestinal tract and their effect on the diversity of intestinal microbial community in fish is less understood. Here, we investigated the intestinal microbial community composition of Atlantic cod after 28 days of exposure to crude oil (concentration range 0.0-0.1 mg/L). RESULTS: Analysis of PAH metabolites in bile samples confirmed that uptake and biotransformation of oil compounds occurred as a result of the exposure. Various evidence for altered microbial communities was found in fish exposed to high (0.1 mg/L) and medium (0.05 mg/L) concentrations of oil when compared to fish exposed to low oil concentration (0.01 mg/L) or no oil (control). First, altered banding patterns were observed on denaturing gradient gel electrophoresis for samples pooled from each treatment group. Secondly, based on 16S rRNA sequences, higher levels of oil exposure were associated with a loss of overall diversity of the gut microbial communities. Furthermore, 8 operational taxonomic units (OTUs) were found to have significantly different relative abundances in samples from fishes exposed to high and medium oil concentrations when compared to samples from the control group and low oil concentration. Among these, only one OTU, a Deferribacterales, had increased relative abundance in samples from fish exposed to high oil concentration. CONCLUSIONS: The results presented herein contribute to a better understanding of the effects of oil contamination on the gut microbial community changes in fish and highlight the importance of further studies into the area. Our findings suggest that increased relative abundance of bacteria belonging to the order Deferribacterales may be indicative of exposure to oil at concentrations higher than 0.05 mg/L.

Microbial Community Composition of Tap Water and Biofilms Treated with or without Copper-Silver Ionization.

Copper-silver ionization (CSI) is an in-house water disinfection method primarily installed to eradicate Legionella bacteria from drinking water distribution systems (DWDS). Its effect on the abundance of culturable Legionella and Legionella infections has been documented in several studies. However, the effect of CSI on other bacteria in DWDS is largely unknown. To investigate these effects, we characterized drinking water and biofilm communities in a hospital using CSI, in a neighboring building without CSI, and in treated drinking water at the local water treatment plant. We used 16S rDNA amplicon sequencing and Legionella culturing. The sequencing results revealed three distinct water groups: (1) cold-water samples (no CSI), (2) warm-water samples at the research institute (no CSI), and (3) warm-water samples at the hospital (after CSI; ANOSIM, p < 0.001). Differences between the biofilm communities exposed and not exposed to CSI were less clear (ANOSIM, p = 0.022). No Legionella were cultured, but limited numbers of Legionella sequences were recovered from all 25 water samples (0.2-1.4% relative abundance). The clustering pattern indicated local selection of Legionella types (Kruskal-Wallis, p < 0.001). Furthermore, one unclassified Betaproteobacteria OTU was highly enriched in CSI-treated warm water samples at the hospital (Kruskal-Wallis, p < 0.001).

Temporal dynamics in microbial soil communities at anthrax carcass sites.

BACKGROUND: Anthrax is a globally distributed disease affecting primarily herbivorous mammals. It is caused by the soil-dwelling and spore-forming bacterium Bacillus anthracis. The dormant B. anthracis spores become vegetative after ingestion by grazing mammals. After killing the host, B. anthracis cells return to the soil where they sporulate, completing the lifecycle of the bacterium. Here we present the first study describing temporal microbial soil community changes in Etosha National Park, Namibia, after decomposition of two plains zebra (Equus quagga) anthrax carcasses. To circumvent state-associated-challenges (i.e. vegetative cells/spores) we monitored B. anthracis throughout the period using cultivation, qPCR and shotgun metagenomic sequencing. RESULTS: The combined results suggest that abundance estimation of spore-forming bacteria in their natural habitat by DNA-based approaches alone is insufficient due to poor recovery of DNA from spores. However, our combined approached allowed us to follow B. anthracis population dynamics (vegetative cells and spores) in the soil, along with closely related organisms from the B. cereus group, despite their high sequence similarity. Vegetative B. anthracis abundance peaked early in the time-series and then dropped when cells either sporulated or died. The time-series revealed that after carcass deposition, the typical semi-arid soil community (e.g. Frankiales and Rhizobiales species) becomes temporarily dominated by the orders Bacillales and Pseudomonadales, known to contain plant growth-promoting species. CONCLUSION: Our work indicates that complementing DNA based approaches with cultivation may give a more complete picture of the ecology of spore forming pathogens. Furthermore, the results suggests that the increased vegetation biomass production found at carcass sites is due to both added nutrients and the proliferation of microbial taxa that can be beneficial for plant growth. Thus, future B. anthracis transmission events at carcass sites may be indirectly facilitated by the recruitment of plant-beneficial bacteria.

Genomic analyses suggest parallel ecological divergence in Heliosperma pusillum (Caryophyllaceae).

The mosaic distribution of interbreeding taxa with contrasting ecology and morphology offers an opportunity to study microevolutionary dynamics during ecological divergence. We investigate here the evolutionary history of an alpine and a montane ecotype of Heliosperma pusillum (Caryophyllaceae) in the south-eastern Alps. From six pairs of geographically close populations of the two ecotypes (120 individuals) we obtained a high-coverage restriction site associated DNA sequencing (RADseq) dataset that was used for demographic inference to test the hypothesis of parallel evolution of the two ecotypes. The data are consistent with repeated ecological divergence in H. pusillum, uncovering up to five polytopic origins of one ecotype from the other. A complex evolutionary history is evidenced, with local isolation-with-migration in two population pairs and intra-ecotype migration in two others. In all cases, the time of divergence or secondary contact was inferred as postglacial. A metagenomic analysis on exogenous contaminant RAD sequences suggests divergent microbial communities between the ecotypes. The lack of shared genomic regions of high divergence across population pairs illustrates the action of drift and/or local selection in shaping genetic divergence across repeated cases of ecological divergence.

BLASTGrabber: a bioinformatic tool for visualization, analysis and sequence selection of massive BLAST data.

BACKGROUND: Advances in sequencing efficiency have vastly increased the sizes of biological sequence databases, including many thousands of genome-sequenced species. The BLAST algorithm remains the main search engine for retrieving sequence information, and must consequently handle data on an unprecedented scale. This has been possible due to high-performance computers and parallel processing. However, the raw BLAST output from contemporary searches involving thousands of queries becomes ill-suited for direct human processing. Few programs attempt to directly visualize and interpret BLAST output; those that do often provide a mere basic structuring of BLAST data. RESULTS: Here we present a bioinformatics application named BLASTGrabber suitable for high-throughput sequencing analysis. BLASTGrabber, being implemented as a Java application, is OS-independent and includes a user friendly graphical user interface. Text or XML-formatted BLAST output files can be directly imported, displayed and categorized based on BLAST statistics. Query names and FASTA headers can be analysed by text-mining. In addition to visualizing sequence alignments, BLAST data can be ordered as an interactive taxonomy tree. All modes of analysis support selection, export and storage of data. A Java interface-based plugin structure facilitates the addition of customized third party functionality. CONCLUSION: The BLASTGrabber application introduces new ways of visualizing and analysing massive BLAST output data by integrating taxonomy identification, text mining capabilities and generic multi-dimensional rendering of BLAST hits. The program aims at a non-expert audience in terms of computer skills; the combination of new functionalities makes the program flexible and useful for a broad range of operations.

Detection of Klebsiella pneumoniae in healthy poultry: Insights and perspectives from culturing and metagenomics.

Previously, Klebsiella pneumoniae was found to occur more frequently in healthy turkey flocks than in healthy broiler flocks in Norway. This study aimed to investigate whether this higher occurrence could be attributed to a greater abundance of K. pneumoniae in turkey flocks. We compared culturing, qPCR, and shotgun metagenomic sequencing for the detection and quantification of K. pneumoniae. Using qPCR, we found that 20.7% of broiler flock samples and 63.9% of turkey flock samples were positive for K. pneumoniae. Culturing revealed a significantly higher abundance of K. pneumoniae in turkey flocks compared to broiler flocks. However, metagenomic analysis showed no difference in the relative abundance of Klebsiella spp. between broiler and turkey flocks, and no correlation between the results of culturing and metagenomic quantification. Interestingly, the differential abundance of K. quasipneumoniae was significantly different between the two hosts. Our results indicate that Klebsiella spp. are present in both turkey and broiler flocks at relatively low levels but with a higher abundance in turkey flocks. Our findings also suggest that shotgun metagenomic studies targeting low-abundance taxa such as Klebsiella have poor sensitivity when comparing groups, indicating that reliance on results from metagenomic analysis without experimental validation should be done with caution.

Defining a metagenomic threshold for detecting low abundances of Providencia alcalifaciens in canine faecal samples.

Introduction: Acute haemorrhagic diarrhoea syndrome (AHDS) in dogs is a condition of unknown aetiology. Providencia alcalifaciens is suspected to play a role in the disease as it was commonly found in dogs suffering from AHDS during a Norwegian outbreak in 2019. The role of this bacterium as a constituent of the canine gut microbiota is unknown, hence this study set out to investigate its occurrence in healthy dogs using metagenomics. Materials and methods: To decrease the likelihood of false detection, we established a metagenomic threshold for P. alcalifaciens by spiking culture-negative stool samples with a range of bacterial dilutions and analysing these by qPCR and shotgun metagenomics. The detection limit for P. alcalifaciens was determined and used to establish a metagenomic threshold. The threshold was validated on naturally contaminated faecal samples with known cultivation status for P. alcalifaciens. Finally, the metagenomic threshold was used to determine the occurrence of P. alcalifaciens in shotgun metagenomic datasets from canine faecal samples (n=362) collected in the HUNT One Health project. Results: The metagenomic assay and qPCR had a detection limit of 1.1x103 CFU P. alcalifaciens per faecal sample, which corresponded to a Cq value of 31.4 and 569 unique k-mer counts by shotgun metagenomics. Applying this metagenomic threshold to 362 faecal metagenomic datasets from healthy dogs, P. alcalifaciens was found in only 1.1% (95% CI [0.0, 6.8]) of the samples, and then in low relative abundances (median: 0.04%; range: 0.00 to 0.81%). The sensitivity of the qPCR and shotgun metagenomics assay was low, as only 40% of culture-positive samples were also positive by qPCR and metagenomics. Discussion: Using our detection limit, the occurrence of P. alcalifaciens in faecal samples from healthy dogs was low. Given the low sensitivity of the metagenomic assay, these results do not rule out a significantly higher occurrence of this bacterium at a lower abundance.

Next generation sequencing shows high variation of the intestinal microbial species composition in Atlantic cod caught at a single location.

BACKGROUND: The observation that specific members of the microbial intestinal community can be shared among vertebrate hosts has promoted the concept of a core microbiota whose composition is determined by host-specific selection. Most studies investigating this concept in individual hosts have focused on mammals, yet the diversity of fish lineages provides unique comparative opportunities from an evolutionary, immunological and environmental perspective. Here we describe microbial intestinal communities of eleven individual Atlantic cod (Gadus morhua) caught at a single location based on an extensively 454 sequenced 16S rRNA library of the V3 region. RESULTS: We obtained a total of 280447 sequences and identify 573 Operational Taxonomic Units (OTUs) at 97% sequence similarity level, ranging from 40 to 228 OTUs per individual. We find that ten OTUs are shared, though the number of reads of these OTUs is highly variable. This variation is further illustrated by community diversity estimates that fluctuate several orders of magnitude among specimens. The shared OTUs belong to the orders of Vibrionales, which quantitatively dominate the Atlantic cod intestinal microbiota, followed by variable numbers of Bacteroidales, Erysipelotrichales, Clostridiales, Alteromonadales and Deferribacterales. CONCLUSIONS: The microbial intestinal community composition varies significantly in individual Atlantic cod specimens caught at a single location. This high variation among specimens suggests that a complex combination of factors influence the species distribution of these intestinal communities.

Heterogeneous molecular behavior in liver tumors (HCC and CCA) of two patients with acute intermittent porphyria.

INTRODUCTION: Acute intermittent porphyria (AIP) is a very rare (orphan) metabolic disorder of porphyrin biosynthesis which is characterized by elevated plasma and urine levels of 5-aminolevulinic acid (5-ALA) and porphobilinogen (PBG). Patients with this disorder which is caused by a germline mutation of the hydroxymethylbilan-synthase (HMBS)-gene have a high risk of primary liver cancer which may be determined by disease activity. The exact mechanism of carcinogenesis of this rare tumor is unknown, however. MATERIALS AND METHODS: We analyzed paraffin-embedded formalin-fixed liver tumor and normal liver specimens of two female AIP patients treated at the Munich EPNET center. One patient had developed hepatocellular carcinoma (HCC), the other intrahepatic cholangiocarcinoma (CCA). Since biallelic inactivation of HMBS had been observed in one study, we used Sanger and next-generation sequencing with a 8 gene porphyria panel plus 6 potential modifier loci to search for mutations in DNA extractions. RESULTS: In the patient with the HCC, we found a second inactivating mutation in the HMBS gene in the tumor but not in the adjacent normal liver tissue. No mutation could be found in the liver tissues of the patient with CCA, however. CONCLUSIONS: Biallelic inactivation of HMBS or protoporphyrinogen-oxidase (PPOX), another enzyme of porphyrin biosynthesis, has been observed in patients with acute porphyrias and liver tumors. We could confirm this in our patient with HCC with a mutation in HMBS but not in the one with CCA. Since 5-ALA can be converted into carcinogenic substances such as 4,5-dioxovaleric acid (DOVA) or 3,6-dihydropyrazine-2,5-dipropanoic acid (= cyclic dimerization product of 5-ALA), local production of these metabolites in hepatic areas with complete loss of HMBS activity may contribute to liver carcinogenesis.

Metagenomic and geochemical characterization of pockmarked sediments overlaying the Troll petroleum reservoir in the North Sea.

BACKGROUND: Pockmarks (depressions in the seabed) have been discovered throughout the world's oceans and are often related to hydrocarbon seepage. Although high concentrations of pockmarks are present in the seabed overlaying the Troll oil and gas reservoir in the northern North Sea, geological surveys have not detected hydrocarbon seepage in this area at the present time. In this study we have used metagenomics to characterize the prokaryotic communities inhabiting the surface sediments in the Troll area in relation to geochemical parameters, particularly related to hydrocarbon presence. We also investigated the possibility of increased potential for methane oxidation related to the pockmarks. Five metagenomes from pockmarks and plain seabed sediments were sequenced by pyrosequencing (Roche/454) technology. In addition, two metagenomes from seabed sediments geologically unlikely to be influenced by hydrocarbon seepage (the Oslofjord) were included. The taxonomic distribution and metabolic potential of the metagenomes were analyzed by multivariate analysis and statistical comparisons to reveal variation within and between the two sampling areas. RESULTS: The main difference identified between the two sampling areas was an overabundance of predominantly autotrophic nitrifiers, especially Nitrosopumilus, and oligotrophic marine Gammaproteobacteria in the Troll metagenomes compared to the Oslofjord. Increased potential for degradation of hydrocarbons, especially aromatic hydrocarbons, was detected in two of the Troll samples: one pockmark sample and one from the plain seabed. Although presence of methanotrophic organisms was indicated in all samples, no overabundance in pockmark samples compared to the Oslofjord samples supports no, or only low level, methane seepage in the Troll pockmarks at the present time. CONCLUSIONS: Given the relatively low content of total organic carbon and great depths of hydrocarbon containing sediments in the Troll area, it is possible that at least part of the carbon source available for the predominantly autotrophic nitrifiers thriving in this area originates from sequential prokaryotic degradation and oxidation of hydrocarbons to CO2. By turning CO2 back into organic carbon this subcommunity could play an important environmental role in these dark oligotrophic sediments. The oxidation of ammonia to nitrite and nitrate in this process could further increase the supply of terminal electron acceptors for hydrocarbon degradation.

Closed Genome Sequences of Providencia alcalifaciens Isolates from Dogs.

Eight Providencia alcalifaciens isolates from eight different dogs in Norway with acute hemorrhagic diarrhea were sequenced. Based on Illumina and Oxford Nanopore Technologies sequencing, all of the genomes were complete and closed after hybrid assembly.

A metagenomic study of methanotrophic microorganisms in Coal Oil Point seep sediments.

BACKGROUND: Methane oxidizing prokaryotes in marine sediments are believed to function as a methane filter reducing the oceanic contribution to the global methane emission. In the anoxic parts of the sediments, oxidation of methane is accomplished by anaerobic methanotrophic archaea (ANME) living in syntrophy with sulphate reducing bacteria. This anaerobic oxidation of methane is assumed to be a coupling of reversed methanogenesis and dissimilatory sulphate reduction. Where oxygen is available aerobic methanotrophs take part in methane oxidation. In this study, we used metagenomics to characterize the taxonomic and metabolic potential for methane oxidation at the Tonya seep in the Coal Oil Point area, California. Two metagenomes from different sediment depth horizons (0-4 cm and 10-15 cm below sea floor) were sequenced by 454 technology. The metagenomes were analysed to characterize the distribution of aerobic and anaerobic methanotrophic taxa at the two sediment depths. To gain insight into the metabolic potential the metagenomes were searched for marker genes associated with methane oxidation. RESULTS: Blast searches followed by taxonomic binning in MEGAN revealed aerobic methanotrophs of the genus Methylococcus to be overrepresented in the 0-4 cm metagenome compared to the 10-15 cm metagenome. In the 10-15 cm metagenome, ANME of the ANME-1 clade, were identified as the most abundant methanotrophic taxon with 8.6% of the reads. Searches for particulate methane monooxygenase (pmoA) and methyl-coenzyme M reductase (mcrA), marker genes for aerobic and anaerobic oxidation of methane respectively, identified pmoA in the 0-4 cm metagenome as Methylococcaceae related. The mcrA reads from the 10-15 cm horizon were all classified as originating from the ANME-1 clade. CONCLUSIONS: Most of the taxa detected were present in both metagenomes and differences in community structure and corresponding metabolic potential between the two samples were mainly due to abundance differences. The results suggests that the Tonya Seep sediment is a robust methane filter, where taxa presently dominating this process could be replaced by less abundant methanotrophic taxa in case of changed environmental conditions.

Changes in the fecal microbiota in dogs with acute hemorrhagic diarrhea during an outbreak in Norway.

BACKGROUND: A severe form of acute hemorrhagic diarrhea syndrome (AHDS) occurred in dogs in the Oslo region of Norway during autumn 2019. OBJECTIVES: To characterize the fecal microbiota of dogs with AHDS during the outbreak and compare it to that of healthy dogs from the same period and before the outbreak. ANIMALS: Dogs with AHDS (n = 50), dogs with nonhemorrhagic diarrhea (n = 3), and healthy dogs (n = 11) were sampled during the outbreak. In addition, 78 healthy dogs from the same region were sampled before the outbreak between 2017 and 2018. METHODS: Retrospective case-control study. The fecal microbiotas were characterized using 16S rRNA gene amplicon sequencing. RESULTS: Dogs with AHDS had significantly different microbiota composition (R2  = .07, P < .001) and decreased intestinal diversity relative to healthy dogs from the outbreak period (median, 2.7; range, 0.9-3.5 vs median, 3.2; range, 2.6-4.0; P < .001). The microbiota in dogs with AHDS was characterized by a decrease of Firmicutes and an outgrowth of Proteobacteria, with increased numbers of Clostridium perfringens and Providencia spp. Among the Providencia spp., 1 showed 100% sequence identity with a Providencia alcalifaciens strain that was cultivated and isolated from the same outbreak. No Providencia spp. was found in healthy dogs sampled before the outbreak. CONCLUSIONS AND CLINICAL IMPORTANCE: Dogs with AHDS had marked changes in fecal microbiota including increased numbers of Providencia spp. and C. perfringens, which may have contributed to the severity of this illness.