New avenues for potentially seeking microbial responses to climate change beneath Antarctic ice shelves.

The signs of climate change are undeniable, and the impact of these changes on ecosystem function heavily depends on the response of microbes that underpin the food web. Antarctic ice shelf is a massive mass of floating ice that extends from the continent into the ocean, exerting a profound influence on global carbon cycles. Beneath Antarctic ice shelves, marine ice stores valuable genetic information, where marine microbial communities before the industrial revolution are archived. Here, in this proof-of-concept, by employing a combination of single-cell technologiesand metagenomics, we have been able to sequence frozen microbial DNA (≈300 years old) stored in the marine ice core B15 collected from the Filchnner-Ronne Ice Shelf. Metagenomic data indicated that Proteobacteria and Thaumarchaeota (e.g., Nitrosopumilus spp.), followed by Actinobacteria (e.g., Actinomarinales), were abundant. Remarkably, our data allow us to "travel to the past" and calibrate genomic and genetic evolutionary changes for ecologically relevant microbes and functions, such as Nitrosopumilus spp., preserved in the marine ice (≈300 years old) with those collected recently in seawater under an ice shelf (year 2017). The evolutionary divergence for the ammonia monooxygenase gene amoA involved in chemolithoautotrophy was about 0.88 amino acid and 2.8 nucleotide substitution rate per 100 sites in a century, while the accumulated rate of genomic SNPs was 2,467 per 1 Mb of genome and 100 years. Whether these evolutionary changes remained constant over the last 300 years or accelerated during post-industrial periods remains an open question that will be further elucidated. IMPORTANCE: Several efforts have been undertaken to predict the response of microbes under climate change, mainly based on short-term microcosm experiments under forced conditions. A common concern is that manipulative experiments cannot properly simulate the response of microbes to climate change, which is a long-term evolutionary process. In this proof-of-concept study with a limited sample size, we demonstrate a novel approach yet to be fully explored in science for accessing genetic information from putative past marine microbes preserved under Antarctic ice shelves before the industrial revolution. This potentially allows us estimating evolutionary changes as exemplified in our study. We advocate for gathering a more comprehensive Antarctic marine ice core data sets across various periods and sites. Such a data set would enable the establishment of a robust baseline, facilitating a better assessment of the potential effects of climate change on key genetic signatures of microbes.

Retinitis pigmentosa is associated with shifts in the gut microbiome.

The gut microbiome is known to influence the pathogenesis and progression of neurodegenerative diseases. However, there has been relatively little focus upon the implications of the gut microbiome in retinal diseases such as retinitis pigmentosa (RP). Here, we investigated changes in gut microbiome composition linked to RP, by assessing both retinal degeneration and gut microbiome in the rd10 mouse model of RP as compared to control C57BL/6J mice. In rd10 mice, retinal responsiveness to flashlight stimuli and visual acuity were deteriorated with respect to observed in age-matched control mice. This functional decline in dystrophic animals was accompanied by photoreceptor loss, morphologic anomalies in photoreceptor cells and retinal reactive gliosis. Furthermore, 16S rRNA gene amplicon sequencing data showed a microbial gut dysbiosis with differences in alpha and beta diversity at the genera, species and amplicon sequence variants (ASV) levels between dystrophic and control mice. Remarkably, four fairly common ASV in healthy gut microbiome belonging to Rikenella spp., Muribaculaceace spp., Prevotellaceae UCG-001 spp., and Bacilli spp. were absent in the gut microbiome of retinal disease mice, while Bacteroides caecimuris was significantly enriched in mice with RP. The results indicate that retinal degenerative changes in RP are linked to relevant gut microbiome changes. The findings suggest that microbiome shifting could be considered as potential biomarker and therapeutic target for retinal degenerative diseases.

Cationic Dendrimer G2-S16 Inhibits Herpes Simplex Type 2 Infection and Protects Mice Vaginal Microbiome.

The G2-S16 polyanionic carbosilane dendrimer is a promising microbicide that inhibits HSV-2 infection in vitro and in vivo in mice models. This G2-S16 dendrimer inhibits HSV-2 infection even in the presence of semen. Murine models, such as BALB/c female mice, are generally used to characterize host-pathogen interactions within the vaginal tract. However, the composition of endogenous vaginal flora remains largely undefined with modern microbiome analyses. It is important to note that the G2-S16 dendrimer does not change healthy mouse vaginal microbiome where Pseudomonas (10.2-79.1%) and Janthinobacterium (0.7-13%) are the more abundant genera. The HSV-2 vaginally infected female mice showed a significant microbiome alteration because an increase of Staphylococcus (up to 98.8%) and Escherichia (30.76%) levels were observed becoming these bacteria the predominant genera. BALB/c female mice vaginally-treated with the G2-S16 dendrimer and infected with the HSV-2 maintained a healthy vaginal microbiome similar to uninfected female mice. Summarizing, the G2-S16 polyanionic carbosilane dendrimer inhibits the HSV-2 infection in the presence of semen and prevents the alteration of mice female vaginal microbiome.

High-Throughput 16S rRNA Sequencing to Assess Potentially Active Bacteria and Foodborne Pathogens: A Case Example in Ready-to-Eat Food.

Technologies to detect the entire bacterial diversity spectra and foodborne pathogens in food represent a fundamental advantage in the control of foodborne illness. Here, we applied high-throughput 16S rRNA sequencing of amplicons obtained by PCR and RT-PCR from extracted DNA and RNA targeting the entire bacterial community and the active bacterial fraction present in some of the most consumed and distributed ready-to-eat (RTE) salad brands in Europe. Customer demands for RTE food are increasing worldwide along with the number of associated foodborne illness and outbreaks. The total aerobic bacterial count in the analyzed samples was in the range of 2-4 × 106 CFU/g (SD ± 1.54 × 106). Culture validated methods did not detect Salmonella spp., Escherichia coli, and other fecal coliforms. 16S rRNA gene Illumina next-generation sequencing (NGS) data were congruent with these culture-based results and confirmed that these and other well-known foodborne bacterial pathogens, such as Listeria, were not detected. However, the fine-resolution of the NGS method unveiled the presence of the opportunistic pathogens Aeromonas hydrophyla and Rahnella aquatilis (relative frequency of 1.33-7.33%) that were metabolically active in addition to non-pathogenic, active members of Yersinia spp. (relative frequency of 0.0015-0.003%). The common ail and foxA marker genes of Yersinia enterocolitica were not detected by qPCR. Finally, our NGS data identified to non-pathogenic Pseudomonas spp. as the most abundant and metabolically active bacteria in the analyzed RTE salads (53-75% of bacterial abundance). Our data demonstrate the power of sequencing, in parallel, both 16S rRNA and rDNA to identify and discriminate those potentially and metabolically active bacteria and pathogens to provide a more complete view that facilitates the control of foodborne diseases, although further work should be conducted to determine the sensitivity of this method for targeting bacteria.

Insights into the antibiotic resistance dissemination in a wastewater effluent microbiome: bacteria, viruses and vesicles matter.

Wastewater treatment plants effluents are considered as hotspots for the dispersion of antibiotic resistance genes (ARGs) into natural ecosystems. The bacterial resistome (ARG collection in a metagenome) analyses have provided clues on antibacterial resistance dynamics. However, viruses and vesicles are frequently ignored. Here, we addressed the bacterial, viral and vesicle resistomes from a representative wastewater effluent in natural conditions and amended with polymyxin, which is used as a last resort antibiotic. Metagenomics showed that the natural prokaryotic resistome was vast (9000 ARG hits/Gb metagenome) and diverse, while viral resistome was two orders of magnitude lower (50 ARG hits/Gb metagenome) suggesting that viruses rarely encoded ARGs. After polymyxin amendment, data showed no ARG enrichment - including to polymyxin - in the microbiome. Remarkably, microbiomes responded to polymyxin with a vast release of putative vesicles (threefold increase compared with the control), which might be used as 'traps' to decrease the antibiotic concentration. Intriguingly, although polymyxin resistance genes (PRGs) were rare in the microbiome (0.018% of total ARG found), in the viral and vesicle fractions, PRGs were more abundant (0.5%-0.8% of total ARG found). Our data suggest that vesicles could have a more active role in the context of transmission of antibiotic resistances.

Droplet Digital PCR for Estimating Absolute Abundances of Widespread Pelagibacter Viruses.

Absolute abundances of prokaryotes are typically determined by FISH. Due to the lack of a universal conserved gene among all viruses, metagenomic fragment recruitment is commonly used to estimate the relative viral abundance. However, the paucity of absolute virus abundance data hinders our ability to fully understand how viruses drive global microbial populations. The cosmopolitan marine Pelagibacter ubique is host for the highly widespread HTVC010P pelagiphage isolate and the extremely abundant uncultured virus vSAG 37-F6 recently discovered by single-virus genomics. Here we applied droplet digital PCR (ddPCR) to calculate the absolute abundance of these pelagiphage genotypes in the Mediterranean Sea and the Gulf of Maine. Abundances were between 360 and 8,510 virus mL-1 and 1,270-14,400 virus mL-1 for vSAG 37-F6 and HTVC010P, respectively. Illumina PCR-amplicon sequencing corroborated the absence of ddPCR non-specific amplifications for vSAG 37-F6, but showed an overestimation of 6% for HTVC010P from off-targets, genetically unrelated viruses. Absolute abundances of both pelagiphages, two of the most abundance marine viruses, suggest a large viral pelagiphage diversity in marine environments, and show the efficiency and power of ddPCR to disentangle the structure of marine viral communities. Results also highlight the need for a standardized workflow to obtain accurate quantification that allows cross data comparison.

Single-cell genomics uncover Pelagibacter as the putative host of the extremely abundant uncultured 37-F6 viral population in the ocean.

The identification of relevant virus-host pairs that globally account for a large pool of carbon and nutrients in the ocean is paramount to build accurate ecological models. A previous work using single-virus genomics led to the discovery of the uncultured single-virus vSAG 37-F6, originally sorted from the Mediterranean Sea (Blanes Bay Microbial Observatory), that represents one of the most abundant dsDNA viral population in the marine surface virosphere. Here, from same sampling site, we report that a Pelagibacter single-cell contained a viral member of vSAG 37-F6 population, by means of PCR screening of sorted, genome-amplified single cells with vSAG 37-F6-specific primers and whole-genome sequencing. Furthermore, viruses from this population were also found in three other Pelagibacter single cells from the South Pacific and Atlantic oceans. These new uncultured pelagiphages were genetically different from the previously characterized pelagiphage isolates. Data showed that the uncultured vSAG 37-F6 population represents the Pelagibacter phages that inhabit the sunlit ocean better, and contains a vast unrecognized microdiversity.

Deciphering the Human Virome with Single-Virus Genomics and Metagenomics.

Single-cell genomics has unveiled the metabolic potential of dominant microbes inhabiting different environments, including the human body. The lack of genomic information for predominant microbes of the human body, such as bacteriophages, hinders our ability to answer fundamental questions about our viral communities. Here, we applied single-virus genomics (SVGs) to natural human salivary samples in combination with viral metagenomics to gain some insights into the viral community structure of the oral cavity. Saliva samples were processed for viral metagenomics (n = 15) and SVGs (n = 3). A total of 1328 uncultured single viruses were sorted by fluorescence-activated virus sorting followed by whole genome amplification. Sequencing of 24 viral single amplified genomes (vSAGs) showed that half of the vSAGs contained viral hallmark genes. Among those bona fide viruses, the uncultured single virus 92-C13 putatively infecting oral Streptococcus-like species was within the top ≈10 most abundant viruses in the oral virome. Viral gene network and viral metagenomics analyses of 439 oral viruses from cultures, metagenomics, and SVGs revealed that salivary viruses were tentatively structured into ≈200 major viral clusters, corresponding to approximately genus-level groupings. Data showed that none of the publicly available viral isolates, excepting an Actinomyces phage, were significantly abundant in the oral viromes. In addition, none of the obtained viral contigs and vSAGs from this study were present in all viromes. Overall, the data demonstrates that most viral isolates are not naturally abundant in saliva, and furthermore, the predominant viruses in the oral cavity are yet uncharacterized. Results suggest a variable, complex, and interpersonal viral profile. Finally, we demonstrated the power of SVGs in combination with viral metagenomics to unveil the genetic information of the uncultured viruses of the human virome.

Single-virus genomics reveals hidden cosmopolitan and abundant viruses.

Microbes drive ecosystems under constraints imposed by viruses. However, a lack of virus genome information hinders our ability to answer fundamental, biological questions concerning microbial communities. Here we apply single-virus genomics (SVGs) to assess whether portions of marine viral communities are missed by current techniques. The majority of the here-identified 44 viral single-amplified genomes (vSAGs) are more abundant in global ocean virome data sets than published metagenome-assembled viral genomes or isolates. This indicates that vSAGs likely best represent the dsDNA viral populations dominating the oceans. Species-specific recruitment patterns and virome simulation data suggest that vSAGs are highly microdiverse and that microdiversity hinders the metagenomic assembly, which could explain why their genomes have not been identified before. Altogether, SVGs enable the discovery of some of the likely most abundant and ecologically relevant marine viral species, such as vSAG 37-F6, which were overlooked by other methodologies.

Capturing single cell genomes of active polysaccharide degraders: an unexpected contribution of Verrucomicrobia.

Microbial hydrolysis of polysaccharides is critical to ecosystem functioning and is of great interest in diverse biotechnological applications, such as biofuel production and bioremediation. Here we demonstrate the use of a new, efficient approach to recover genomes of active polysaccharide degraders from natural, complex microbial assemblages, using a combination of fluorescently labeled substrates, fluorescence-activated cell sorting, and single cell genomics. We employed this approach to analyze freshwater and coastal bacterioplankton for degraders of laminarin and xylan, two of the most abundant storage and structural polysaccharides in nature. Our results suggest that a few phylotypes of Verrucomicrobia make a considerable contribution to polysaccharide degradation, although they constituted only a minor fraction of the total microbial community. Genomic sequencing of five cells, representing the most predominant, polysaccharide-active Verrucomicrobia phylotype, revealed significant enrichment in genes encoding a wide spectrum of glycoside hydrolases, sulfatases, peptidases, carbohydrate lyases and esterases, confirming that these organisms were well equipped for the hydrolysis of diverse polysaccharides. Remarkably, this enrichment was on average higher than in the sequenced representatives of Bacteroidetes, which are frequently regarded as highly efficient biopolymer degraders. These findings shed light on the ecological roles of uncultured Verrucomicrobia and suggest specific taxa as promising bioprospecting targets. The employed method offers a powerful tool to rapidly identify and recover discrete genomes of active players in polysaccharide degradation, without the need for cultivation.