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Specialized metabolites produced by microorganisms found in ocean sediments display a wide range of clinically relevant bioactivities, including antimicrobial, anticancer, antiviral, and anti-inflammatory. Due to limitations in our ability to culture many benthic microorganisms under laboratory conditions, their potential to produce bioactive compounds remains underexplored. However, the advent of modern mass spectrometry technologies and data analysis methods for chemical structure prediction has aided in the discovery of such metabolites from complex mixtures. In this study, ocean sediments were collected from Baffin Bay (Canadian Arctic) and the Gulf of Maine for untargeted metabolomics using mass spectrometry. A direct examination of prepared organic extracts identified 1468 spectra, of which ∼45% could be annotated using in silico analysis methods. A comparable number of spectral features were detected in sediments collected from both locations, but 16S rRNA gene sequencing revealed a significantly more diverse bacterial community in samples from Baffin Bay. Based on spectral abundance, 12 specialized metabolites known to be associated with bacteria were selected for discussion. The application of metabolomics directly on marine sediments provides an avenue for culture-independent detection of metabolites produced under natural settings. The strategy can help prioritize samples for novel bioactive metabolite discovery using traditional workflows.

Caliciviruses are ssRNA viruses that can infect a wide range of hosts, including birds. While several avian caliciviruses have been discovered, their taxonomy and host distribution are largely unknown. We molecularly characterized a novel calicivirus (trumpeter swan calicivirus: TruSCV) in trumpeter swans over-wintering in south-west British Columbia, Canada. The positivity rate was 20.3% (14/69) and there were no significant differences in infection rates between males (5/34, 14.7%) and females (9/35, 25.7%) or among considered age groups (juveniles: 4/14, 28.6%; sub-adults: 1/9, 11.1%; adults: 9/46, 19.6%). Twelve infected swans died of lead poisoning, one because of starvation, and one from physical injuries. TruSCV complete genome possessed the typical organization and protein motifs of caliciviruses and a type 2 IRES and its closest relative was a virus circulating in Australian ducks. Phylogenetic analyses showed the existence of 34 different but monophyletic avian caliciviruses. These viruses, while having conserved genomic organization and protein motifs, possess different IRES types and group in several divergent clades, with only two of them corresponding to currently defined genera, highlighting the need for epidemiological investigations and systematic analyses to better define their taxonomy. Follow-up studies are needed to elucidate the diversity, distribution, and pathogenic potential of TruSCV.

Circoviruses (genus

Metagenomic methods are powerful tools to investigate viral diversity in biological or environmental samples and to identify previously unknown viruses. We used RNA metagenomics to identify, in the gut of red-backed voles, the nearly complete genomes of two novel members of the

Aquaculture is a rapidly expanding industry and is now one of the primary sources of all consumed seafood. Intensive aquaculture production is associated with organic enrichment, which occurs as organic material settles onto the seafloor, creating anoxic conditions which disrupt ecological processes. Bacteria are sensitive bioindicators of organic enrichment, and supervised classifiers using features derived from 16s rRNA gene sequences have shown potential to become useful in aquaculture environmental monitoring. Current taxonomy-based approaches, however, are time intensive and built upon emergent features which cannot easily be condensed into a monitoring pipeline. Here, we used a taxonomy-free approach to examine 16s rRNA gene sequences derived from flocculent matter underneath and in proximity to hard-bottom salmon aquaculture sites in Newfoundland, Canada. Tetranucleotide frequencies (k = 4) were tabulated from sample sequences and included as features in a machine learning pipeline using the random forest algorithm to predict 4 levels of benthic disturbance; resulting classifications were compared to those obtained using a published taxonomy-based approach. Our results show that k-mer count features can effectively be used to create highly accurate predictions of benthic disturbance and can resolve intermediate changes in seafloor condition. In addition, we present a robust assessment of model performance which accounts for the effect of randomness in model creation. This work outlines a flexible framework for environmental assessments at aquaculture sites that is highly reproducible and free of taxonomy-assignment bias.

The genus Protoparvovirus (family Parvoviridae) includes several viruses of carnivores. We describe a novel fox protoparvovirus, which we named Newlavirus as it was discovered in samples from Newfoundland and Labrador, Canada. Analysis of the full non-structural protein (NS1) sequence indicates that this virus is a previously uncharacterized species. Newlavirus showed high prevalence in foxes from both the mainland (Labrador, 54/137, 39.4%) and the island of Newfoundland (22/50, 44%) but was not detected in samples from other carnivores, including coyotes (n = 92), lynx (n = 58), martens (n = 146), mink (n = 47), ermines (n = 17), dogs (n = 48), and ringed (n = 4), harp (n = 6), bearded (n = 6), and harbor (n = 2) seals. Newlavirus was found at similar rates in stool and spleen (24/80, 30% vs. 59/152, 38.8%, p = 0.2) but at lower rates in lymph nodes (2/37, 5.4%, p &lt, 0.01). Sequencing a fragment of approximately 750 nt of the capsid protein gene from 53 samples showed a high frequency of co-infection by more than one strain (33.9%), high genetic diversity with 13 genotypes with low sequence identities (70.5–87.8%), and no geographic segregation of strains. Given the high prevalence, high diversity, and the lack of identification in other species, foxes are likely the natural reservoir of Newlavirus, and further studies should investigate its distribution.

Parvoviruses are small single-stranded DNA viruses that can infect both vertebrates and invertebrates. We report here the full characterization of novel viruses we identified in ducks, including two viral species within the subfamily Hamaparvovirinae (duck-associated chapparvovirus, DAC) and a novel species within the subfamily Densovirinae (duck-associated ambidensovirus, DAAD). Overall, 5.7% and 21.1% of the 123 screened ducks (American black ducks, mallards, northern pintail) were positive for DAC and DAAD, respectively, and both viruses were more frequently detected in autumn than in winter. Genome organization and predicted transcription profiles of DAC and DAAD were similar to viruses of the genera Chaphamaparvovirus and Protoambidensovirus, respectively. Their association to these genera was also demonstrated by subfamily-wide phylogenetic and distance analyses of non-structural protein NS1 sequences. While DACs were included in a highly supported clade of avian viruses, no definitive conclusions could be drawn about the host type of DAAD because it was phylogenetically close to viruses found in vertebrates and invertebrates and analyses of codon usage bias and nucleotide frequencies of viruses within the family Parvoviridae showed no clear host-based viral segregation. This study highlights the high parvoviral diversity in the avian reservoir with many avian-associated parvoviruses likely yet to be discovered.

High-throughput sequencing (HTS) technologies are becoming increasingly important within microbiology research, but aspects of library preparation, such as high cost per sample or strict input requirements, make HTS difficult to implement in some niche applications and for research groups on a budget. To answer these necessities, we developed ViDiT, a customizable, PCR-based, extremely low-cost (less than US$5 per sample), and versatile library preparation method, and CACTUS, an analysis pipeline designed to rely on cloud computing power to generate high-quality data from ViDiT-based experiments without the need of expensive servers. We demonstrate here the versatility and utility of these methods within three fields of microbiology: virus discovery, amplicon-based viral genome sequencing, and microbiome profiling. ViDiT–CACTUS allowed the identification of viral fragments from 25 different viral families from 36 oropharyngeal–cloacal swabs collected from wild birds, the sequencing of three almost complete genomes of avian influenza A viruses (>90% coverage), and the characterization and functional profiling of the complete microbial diversity (bacteria, archaea, viruses) within a deep-sea carnivorous sponge. ViDiT–CACTUS demonstrated its validity in a wide range of microbiology applications, and its simplicity and modularity make it easily implementable in any molecular biology laboratory, towards various research goals.

While sponges are generally known to host a wide range of microbial associates, the composition and specificity of the microbial communities in carnivorous sponges are poorly understood. We used 16S rRNA gene data to examine and compare the bacterial communities associated with distinct anatomical regions of two carnivorous sponge species, Chondrocladia grandis and Cladorhiza oxeata, sampled from Baffin Bay and the Gulf of Maine (C. grandis only). The two sponge species hosted distinct bacterial communities, with taxonomic diversity being greater in C. grandis. Some bacterial taxa (including particular oligotypes) were consistently recovered in multiple host individuals from geographically distant sites, suggesting specificity. Within C. grandis, certain bacterial taxa were enriched in particular anatomical regions, suggesting functional roles in carnivorous sponge metabolism or other biological processes. Stable isotope analysis provided no evidence for methanotrophy in the sponges examined, but Gulf of Maine C. grandis might incorporate 13C-depleted carbon via the bacteria-mediated heterotrophic degradation of other hydrocarbons. Overall, our results demonstrate that the carnivorous sponge microbiome appears host species specific and stable, even over large geographical areas. The observed differences in bacterial community composition and diversity between C. grandis and C. oxeata may reflect differences in trophic adaptability, specialization, and overall reliance on associated bacteria.

Aquaculture has become a rapidly growing industry: over the past 3 decades, commercial production has steadily increased, and further expansion seems likely. However, the rise of aquaculture has been accompanied by concerns, especially regarding environmental sustainability. Substrates located under aquaculture sites receive large influxes of organic matter that can subsequently create anoxic conditions and thereby impact existing benthic communities. Shifts in the relative abundance of specific groups of bacteria could prove to be important indicators of impact and remediation. Here, we investigated bacterial community composition via 16S rRNA gene sequencing on isolated DNA from flocculent matter samples and associated bacterial mats under a hard-bottom aquaculture site in Newfoundland, Canada. We describe the heterogeneous community present in the flocculent matter, characterized by high relative abundances of the genera Spirochaeta (12%), Prolixibacter (5.6%) and Marinifilum (4.6%). Bacterial mats were not composed of Beggiatoa as often hypothesized, but instead were dominated by the genera Spirochaeta (15%), Prevotella (21%), Meniscus (11%) and Odoribacter (20%). Our findings provide insights into the bacterial composition of flocculent matter deposited on hard substrates and undergoing degradation, and point to 3 unexpected bacterial genera as potential indicators of organic enrichment.

Coastal aquaculture has experienced substantial growth in the last few decades and associated impacts on natural environments are of increasing importance. Understanding both the effects of aquaculture on marine ecosystems and the processes of recovery during fallowing periods is crucial for the development of a more environmentally sustainable industry. Because bacteria are sensitive to environmental change, surveying fluctuations in bacterial communities is a promising tool for monitoring the status of benthic environments. Here, we used 16S rRNA gene high-throughput sequencing to characterize bacterial communities in flocculent matter samples collected over a period of 3 years and at various distances from cages (0–200 meters) at production and fallow (3–35 months) salmon aquaculture sites in southern Newfoundland to evaluate the environmental impact of aquaculture on predominantly hard-bottom substrates. Bacterial composition analysis revealed four clusters, three of which (defined as “recently disturbed,” “intermediate impact,” and “high impact”) differed markedly from a fourth “low impact” cluster that contained far-field samples collected >500 m from cages. Samples within the high impact group were most often collected directly under cages, whereas those in the intermediate impact group were mainly sampled from 20 to 40 m from cages. Large scale phylum shifts (increases of Bacteroidetes, Firmicutes, Spirochaetes, and decreases in Proteobacteria and Epsilonbacteraeota) and a decline in bacterial diversity were observed in the high impact cluster, indicating significant ecological change. Samples from sites of different fallow duration were found in the high impact cluster, indicating a lack of recovery, even after 35 months of fallowing. Finally, we identified 28 genera as bacterial biomarkers, specific to one or more clusters, including genera associated with organically enriched environments and previously reported in the context of aquaculture impacts. Tracking the relative abundance of biomarkers in relation to different lengths of fallowing in the three more impacted clusters showed that these markers remained significantly above low impact cluster levels at all times, further pointing toward incomplete recovery. Our results suggest that coastal aquaculture on hard-bottom substrates is prone to long lasting impacts on bacterial communities, especially below cages, and that effects can be accurately tracked using bacterial community profiles or specific biomarkers.

Lemurs are highly endangered mammals inhabiting the forests of Madagascar. In this study, we performed virus discovery on serum samples collected from 84 wild lemurs and identified viral sequence fragments from 4 novel viruses within the family Flaviviridae, including members of the genera Hepacivirus and Pegivirus. The sifaka hepacivirus (SifHV, two genotypes) and pegivirus (SifPgV, two genotypes) were discovered in the diademed sifaka (Propithecus diadema), while other pegiviral fragments were detected in samples from the indri (Indri indri, IndPgV) and the weasel sportive lemur (Lepilemur mustelinus, LepPgV). Although data are preliminary, each viral species appeared host species-specific and frequent infection was detected (18 of 84 individuals were positive for at least one virus). The complete coding sequence and partial 5’ and 3’ untranslated regions (UTRs) were obtained for SifHV and its genomic organization was consistent with that of other hepaciviruses, with one unique polyprotein and highly structured UTRs. Phylogenetic analyses showed the SifHV belonged to a clade that includes several viral species identified in rodents from Asia and North America, while SifPgV and IndPgV were more closely related to pegiviral species A and C, that include viruses found in humans as well as New- and Old-World monkeys. Our results support the current proposed model of virus-host co-divergence with frequent occurrence of cross-species transmission for these genera and highlight how the discovery of more members of the Flaviviridae can help clarify the ecology and evolutionary history of these viruses. Furthermore, this knowledge is important for conservation and captive management of lemurs.

Coronafacoyl phytotoxins are an important family of plant toxins that are produced by several different phytopathogenic bacteria, including the gammaproteobacterium Pseudomonas syringae and the actinobacterium Streptomyces scabiei (formerly Streptomyces scabies ). The phytotoxins consist of coronafacic acid (CFA) linked via an amide bond to different amino acids or amino acid derivatives. Previous work suggested that S. scabiei and P. syringae use distinct biosynthetic pathways for producing CFA, which is subsequently linked to its amino acid partner to form the complete phytotoxin. Here, we provide further evidence that the S. scabiei CFA biosynthetic pathway is novel by characterizing the role of CYP107AK1, a predicted cytochrome P450 that has no homologue in P. syringae . Deletion of the CYP107AK1 gene abolished production of coronafacoyl-isoleucine (CFA-Ile), the primary coronafacoyl phytotoxin produced by S. scabiei . Structural elucidation of accumulated biosynthetic intermediates in the Δ CYP107AK1 mutant indicated that CYP107AK1 is required for introducing the oxygen atom that ultimately forms the carbonyl group in the CFA backbone. The CYP107AK1 gene along with two additional genes involved in CFA-Ile biosynthesis in S. scabiei were found to be associated with putative CFA biosynthetic genes in other actinobacteria but not in other organisms. Analysis of the overall genetic content and organization of known and putative CFA biosynthetic gene clusters, together with phylogenetic analysis of the core biosynthetic genes, indicates that horizontal gene transfer has played an important role in the dissemination of the gene cluster and that rearrangement, insertion, and/or deletion events have likely contributed to the divergent biosynthetic evolution of coronafacoyl phytotoxins in bacteria. IMPORTANCE The ability of plants to defend themselves against invading pathogens relies on complex signaling pathways that are controlled by key phytohormones such as jasmonic acid (JA). Some phytopathogenic bacteria have evolved the ability to manipulate JA signaling in order to overcome host defenses by producing coronatine (COR), which functions as a potent JA mimic. COR and COR-like molecules, collectively referred to as coronafacoyl phytotoxins, are produced by several different plant-pathogenic bacteria, and this study provides supporting evidence that different biosynthetic pathways are utilized by different bacteria for production of these phytotoxins. In addition, our study provides a greater understanding of how coronafacoyl phytotoxin biosynthesis may have evolved in phylogenetically distinct bacteria, and we demonstrate that production of these compounds may be more widespread than previously recognized and that their role for the producing organism may not be limited to host-pathogen interactions.

Acute central nervous system (CNS) infections cause substantial morbidity and mortality, but the etiology remains unknown in a large proportion of cases. We identified and characterized the full genome of a novel cyclovirus (tentatively named cyclovirus-Vietnam [CyCV-VN]) in cerebrospinal fluid (CSF) specimens of two Vietnamese patients with CNS infections of unknown etiology. CyCV-VN was subsequently detected in 4% of 642 CSF specimens from Vietnamese patients with suspected CNS infections and none of 122 CSFs from patients with noninfectious neurological disorders. Detection rates were similar in patients with CNS infections of unknown etiology and those in whom other pathogens were detected. A similar detection rate in feces from healthy children suggested food-borne or orofecal transmission routes, while high detection rates in feces from pigs and poultry (average, 58%) suggested the existence of animal reservoirs for such transmission. Further research is needed to address the epidemiology and pathogenicity of this novel, potentially zoonotic virus., IMPORTANCE Acute central nervous system (CNS) infections cause substantial morbidity and mortality, but the etiology frequently remains unknown, which hampers development of therapeutic or preventive strategies. Hence, identification of novel pathogens is essential and is facilitated by current next-generation sequencing-based methods. Using such technology, we identified and characterized the full genome of a novel cyclovirus in cerebrospinal fluid (CSF) specimens from two Vietnamese patients with CNS infections of unknown etiology, which was subsequently detected in none of 122 CSF specimens from patients with noninfectious neurological disorders but 4% of 642 CSF specimens from Vietnamese patients with suspected or confirmed CNS infections. Similar detection rates in feces from healthy children suggested food-borne or orofecal transmission routes, while frequent detection in feces from Vietnamese pigs and poultry (average, 58%) suggested the existence of animal reservoirs for such transmission. Further studies are needed to address the epidemiology and pathogenicity of this novel, potentially zoonotic virus.

Discovery of new viruses has been boosted by novel deep sequencing technologies. Currently, many viruses can be identified by sequencing without knowledge of the pathogenicity of the virus. However, attributing the presence of a virus in patient material to a disease in the patient can be a challenge. One approach to meet this challenge is identification of viral sequences based on enrichment by autologous patient antibody capture. This method facilitates identification of viruses that have provoked an immune response within the patient and may increase the sensitivity of the current virus discovery techniques. To demonstrate the utility of this method, virus discovery deep sequencing (VIDISCA-454) was performed on clinical samples from 19 patients: 13 with a known respiratory viral infection and 6 with a known gastrointestinal viral infection. Patient sera was collected from one to several months after the acute infection phase. Input and antibody capture material was sequenced and enrichment was assessed. In 18 of the 19 patients, viral reads from immunogenic viruses were enriched by antibody capture (ranging between 1.5x to 343x in respiratory material, and 1.4x to 53x in stool). Enriched reads were also determined in an identity independent manner by using a novel algorithm Xcompare. In 16 of the 19 patients, 21% to 100% of the enriched reads were derived from infecting viruses. In conclusion, the technique provides a novel approach to specifically identify immunogenic viral sequences among the bulk of sequences which are usually encountered during virus discovery metagenomics.

Four methods for the species identification of coagulase-negative staphylococci in the medical microbiology laboratory were compared with 444 consecutive isolates. The methods included (i) the reference method based on growth tests, (ii) API ID 32 Staph (bioMérieux), (iii) Staph-Zym (Rosco), and (iv) a rapid 4-h method developed in our laboratory (UZA method). The last method is based on the detection within 4 h of enzymatic activity of heavy bacterial suspensions in three substrate solutions (nongrowth tests). For 16.5% of the isolates some supplementary growth tests read after 24 h had to be added to the enzyme data for satisfactory identification. The reference method failed to identify four isolates. Of the 440 isolates identified by the reference method, API ID 32 Staph, Staph-Zym, and the UZA method correctly identified 419 (95.2%), 429 (97.5%), and 430 (97.7%) and misidentified 8 (1.8%), 4 (0.9%), and 1 (0.2%), respectively. Staphylococcus epidermidis, S. haemolyticus, S. lugdunensis, S. schleiferi, and S. capitis were identified with an accuracy of 98 to 100% by all the systems tested. S. capitis subsp. ureolyticus was not recognized by the API ID 32 system because the biochemical profiles for it are not yet included in the corresponding database. Whereas API ID 32 identified all 13 S. warneri isolates, both Staph-Zym and the UZA method missed 2 of these. S. hominis was identified with the least accuracy by the API ID 32 system (26 of 39 isolates), whereas the UZA and Staph-Zym methods identified 36 of the isolates belonging to this species. The UZA method did not identify any of the S. cohnii, S. xylosus, S. lentus, and S. sciuri strains, since it included no discriminatory tests for these species, because they are extremely rarely found in humans. Of all 440 isolates tested, the UZA method failed to identify 9 and misidentified 1 other. Eighty-one percent of the isolates were identified within 4 h and 97.7% were identified after 24 h, at considerably less expense than by the API ID 32 Staph and Staph-Zym methods.

A commercially available fluorescence test (Crystal MRSA ID System; BBL) for the rapid detection of methicillin resistance in Staphylococcus aureus was evaluated for the detection of methicillin resistance in coagulase-negative staphylococci. The assay was compared with the agar dilution method and mecA detection by PCR. Provided that one uses an inoculum equivalent to a no. 2 McFarland standard instead of a no. 0.5 McFarland standard, which is used for S. aureus, the Crystal MRSA ID System detects methicillin resistance among coagulase-negative staphylococci with sensitivity, specificity, and positive and negative predictive values of 98.5, 95.4, 96.3, and 98.1%, respectively.

We describe a patient with a serious urinary tract infection due to Actinomyces bernardiae (formerly classified as CDC [Centers for Disease Control and Prevention, Atlanta] fermentative coryneform group 2). Our patient had long-standing bladder dysfunction and developed hydroureteronephrosis, renal lithiasis, perirenal abscesses, and septicemia. A. bernardiae is distinct from Actinomyces pyogenes and several CDC coryneform groups.