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Nearly half of the sequenced bacteria are lysogens and many of their prophages encode adaptive traits. Yet, the variables driving prophage distribution remain undetermined. We identified 2246 prophages in complete bacterial genomes to study the genetic and life-history traits associated with lysogeny. While optimal growth temperatures and average cell volumes were not associated with lysogeny, prophages were more frequent in pathogens and in bacteria with small minimal doubling times. Their frequency also increased with genome size, but only for genomes smaller than 6 Mb. The number of spacers in CRISPR-Cas systems and the frequency of type III systems were anticorrelated with prophage frequency, but lysogens were more likely to encode type I and type II systems. The minimal doubling time was the trait most correlated with lysogeny, followed by genome size and pathogenicity. We propose that bacteria with highly variable growth rates often encounter lower opportunity costs for lysogeny relative to lysis. These results contribute to explain the paucity of temperate phages in certain bacterial clades and of bacterial lysogens in certain environments. They suggest that genetic and life-history traits affect the contributions of temperate phages to bacterial genomes.

The gut of healthy human neonates is usually devoid of viruses at birth, but quickly becomes colonized, which—in some cases—leads to gastrointestinal disorders 1 – 4 . Here we show that the assembly of the viral community in neonates takes place in distinct steps. Fluorescent staining of virus-like particles purified from infant meconium or early stool samples shows few or no particles, but by one month of life particle numbers increase to 10 9 per gram, and these numbers seem to persist throughout life 5 – 7 . We investigated the origin of these viral populations using shotgun metagenomic sequencing of virus-enriched preparations and whole microbial communities, followed by targeted microbiological analyses. Results indicate that, early after birth, pioneer bacteria colonize the infant gut and by one month prophages induced from these bacteria provide the predominant population of virus-like particles. By four months of life, identifiable viruses that replicate in human cells become more prominent. Multiple human viruses were more abundant in stool samples from babies who were exclusively fed on formula milk compared with those fed partially or fully on breast milk, paralleling reports that breast milk can be protective against viral infections 8 – 10 . Bacteriophage populations also differed depending on whether or not the infant was breastfed. We show that the colonization of the infant gut is stepwise, first mainly by temperate bacteriophages induced from pioneer bacteria, and later by viruses that replicate in human cells; this second phase is modulated by breastfeeding. The infant gut is colonized first by temperate bacteriophages induced from pioneer bacteria and later by viruses that replicate in human cells, the populations of which are modulated by breastfeeding.

Despite an overall temporal stability in time of the human gut microbiota at the phylum level, strong variations in species abundance have been observed. We are far from a clear understanding of what promotes or disrupts the stability of microbiome communities. Environmental factors, like food or antibiotic use, modify the gut microbiota composition, but their overall impacts remain relatively low. Phages, the viruses that infect bacteria, might constitute important factors explaining temporal variations in species abundance. Gut bacteria harbour numerous prophages, or dormant viruses, which can evolve to become ultravirulent phage mutants, potentially leading to important bacterial death. Whether such phenomenon occurs in the mammal’s microbiota has been largely unexplored. Here we studied temperate phage–bacteria coevolution in gnotoxenic mice colonised with Roseburia intestinalis, a dominant symbiont of the human gut microbiota, and Escherichia coli, a sub-dominant member of the same microbiota. We show that R. intestinalis L1-82 harbours two active prophages, Jekyll and Shimadzu. We observed the systematic evolution in mice of ultravirulent Shimadzu phage mutants, which led to a collapse of R. intestinalis population. In a second step, phage infection drove the fast counterevolution of host phage resistance mainly through phage-derived spacer acquisition in a clustered regularly interspaced short palindromic repeats array. Alternatively, phage resistance was conferred by a prophage originating from an ultravirulent phage with a restored ability to lysogenize. Our results demonstrate that prophages are a potential source of ultravirulent phages that can successfully infect most of the susceptible bacteria. This suggests that prophages can play important roles in the short-term temporal variations observed in the composition of the gut microbiota.

Viral diversity and life cycles are poorly understood in the human gut and other body habitats. Phages and their encoded functions may provide informative signatures of a human microbiota and of microbial community responses to various disturbances, and may indicate whether community health or dysfunction is manifest after apparent recovery from a disease or therapeutic intervention. Here we report sequencing of the viromes (metagenomes) of virus-like particles isolated from faecal samples collected from healthy adult female monozygotic twins and their mothers at three time points over a one-year period. We compared these data sets with data sets of sequenced bacterial 16S ribosomal RNA genes and total-faecal-community DNA. Co-twins and their mothers share a significantly greater degree of similarity in their faecal bacterial communities than do unrelated individuals. In contrast, viromes are unique to individuals regardless of their degree of genetic relatedness. Despite remarkable interpersonal variations in viromes and their encoded functions, intrapersonal diversity is very low, with >95% of virotypes retained over the period surveyed, and with viromes dominated by a few temperate phages that exhibit remarkable genetic stability. These results indicate that a predatory viral–microbial dynamic, manifest in a number of other characterized environmental ecosystems, is notably absent in the very distal intestine. Variations in the gut virome The microbial component of the human gut microbiota has been the focus of much research interest recently. Now another layer of complexity is added in the form of the first viral metagenome, determined from virus-like particles isolated from faecal samples of four sets of identical twins and their mothers. A previous study showed that co-twins and their mothers share a significantly greater degree of similarity in their faecal bacterial communities than do unrelated individuals. By contrast, the viromes are found to be unique to individuals, regardless of their degree of genetic relatedness. And in each individual, there was little change in the viromes during the one-year span of the study. The microbial content of the human gut has been the focus of much research interest recently. Now another layer of complexity has been added: the viral content of the gut. Virus-like particles were isolated from faecal samples from four sets of identical twins and their mothers, at three time points over a one-year period. The viromes (metagenomes) of these particles were then sequenced. The results show that there is high interpersonal variation in viromes, but that intrapersonal diversity was very low over this time period.

Abstract Pseudomonas aeruginosa has increasing clinical relevance and commonly occupies the cystic fibrosis (CF) airways. Its ability to colonize and persist in diverse niches is attributed to its large accessory genome, where prophages represent a common feature and may contribute to its fitness and persistence. We focused on the CF airways niche and used 197 longitudinal isolates from 12 patients persistently infected by P. aeruginosa. We computationally predicted intact prophages for each longitudinal group and scored their long-term persistence. We then confirmed prophage inducibility and mapped their location in the host chromosome with lysate sequencing. Using comparative genomics, we evaluated prophage genomic diversity, long-term persistence, and level of genomic maintenance. Our findings support previous findings that most P. aeruginosa genomes harbour prophages some of which can self-induce, and that a common CF-treating antibiotic, ciprofloxacin, can induce prophages. Induced prophage genomes displayed high diversity and even genomic novelty. Finally, all induced prophages persisted long-term with their genomes avoiding gene loss and degradation over 4 years of host replication in the stressful CF airways niche. This and our detection of phage genes, which contribute to host competitiveness and adaptation, lends support to our hypothesis that the vast majority of prophages detected as intact and inducible in this study facilitated their host fitness and persistence. Clinical Pseudomonas aeruginosa genomes contain an array of intact, inducible prophages, which are genomically highly diverse sometimes novel, and persist long-term in their niche with their genomes virtually unchanged.

Background Phages are critical components of the gut microbiome, influencing bacterial composition and function as predators, parasites, and modulators of bacterial phenotypes. Prophages, integrated forms of these phages, are prevalent in many bacterial genomes and play a role in bacterial adaptation and evolution. However, the diversity and stability of prophages within gut commensals, particularly in the genera Bacteroides and Phocaeicola , remain underexplored. This study aims to screen and characterize prophages in these genera, providing insights into their diversity, host range, and temporal dynamics in the human gut. Results Using a combination of three bioinformatic tools—Cenote-Taker 3, Vibrant, and PHASTER—we conducted a comprehensive analysis of prophages in Bacteroides and Phocaeicola . Cenote-Taker 3 identified the most diverse set of prophages, with significant overlaps observed between the tools. After clustering high-quality prophages, we identified 22 unique viral operational taxonomic units (vOTUs). Notably, comparisons between prophages identified in isolated bacterial genomes, metaviromes, and large public gut virome databases revealed a broader host range than initially observed in single isolates. Certain prophages were consistent across time points and individuals, suggesting temporal stability. All identified prophages belonged to the Caudoviricetes class and contained genes related to antibiotic resistance, toxin production, and metabolic processes. Conclusions The combined use of multiple prophage detection tools allowed for a more comprehensive assessment of prophage diversity in Bacteroides and Phocaeicola . The identified prophages were not only prevalent but also exhibited broad host ranges and temporal stability. The presence of antibiotic resistance and toxin genes suggests that these prophages may significantly influence bacterial community structure and function in the gut, with potential implications for human health. These findings highlight the importance of using diverse detection tools to accurately assess prophage diversity and dynamics.

Although tailed bacteriophages (phages) of the class Caudoviricetes are thought to constitute the most abundant and ecologically relevant group of phages that can integrate their genome into the host chromosome, it is becoming increasingly clear that other prophages are widespread. Here, we show that prophages derived from filamentous and tailless phages with genome sizes below 16 kb make up the majority of prophages in marine bacteria of the genus Vibrio. To estimate prophage prevalence unaffected by database biases, we combined comparative genomics and chemical induction of 58 diverse Vibrio cyclitrophicus isolates, resulting in 107 well-curated prophages. Complemented with computationally predicted prophages, we obtained 1158 prophages from 931 naturally co-existing strains of the family Vibrionaceae. Prophages resembling tailless and filamentous phages predominated, accounting for 80% of all prophages in V. cyclitrophicus and 60% across the Vibrionaceae. In our experimental model, prophages of all three viral realms actively replicated upon induction indicating their ability to transfer to new hosts. Indeed, prophages were rapidly gained and lost, as suggested by variable prophage content between closely related V. cyclitrophicus. Prophages related to filamentous and tailless phages were integrated into only three genomic locations and restored the function of their integration site. Despite their small size, they contained highly diverse accessory genes that may contribute to host fitness, such as phage defense systems. We propose that, like their well-studied tailed equivalent, tailless and filamentous temperate phages are active and highly abundant drivers of host ecology and evolution in marine Vibrio, which have been largely overlooked.

Background Staphylococcal infections, caused by a large variety of species within the Staphylococcus genus, are a threat to human health. Although antibiotics are the current choice of treatment for these infections, bacteriophage (phage) therapy has been used with success against Staphylococcus since the dawn of phage therapy. In 2020 a new coagulase-negative species named Staphylococcus borealis was described in Norway. In this study, we focused on understanding phage infections in S. borealis . Methods: First, we predict the presence of prophages and phage-defence mechanisms in the genomes of a collection of twelve S. borealis strains by bioinformatics. We also attempted to isolate S. borealis -infecting phages from Norwegian samples and tested the host-range of three known staphylococcal phages against a panel of fifty Norwegian staphylococcal strains. Results: The presence of prophages and phage defence systems was verified in all tested S. borealis strains. No local Norwegian phages could be obtained in a phage isolation attempt targeted towards S. borealis . The host range analysis shows that phage ISP, originally isolated in the 1920s and still used for phage therapy to date, can infect S. capitis and the S. borealis Hus23 strain. Phage ISP was shown to limit S. borealis Hus23 growth in liquid cultures and lower the formation of biofilm by the bacterium. The efficiency of plating of phage ISP can be improved by repeated passages in the new S. borealis Hus23 host. Conclusions: here we expand the known host range of the traditional phage ISP by showing that it also infects S. borealis and can be adapted to the new host by serial passages, showcasing the flexibility of phages as an antimicrobial strategy.

Filamentous phages (FPs) have been recently isolated from Acinetobacter baumannii . While FPs are known to modulate the virulence of some Gram-negative pathogens, their role in A. baumannii has not been fully explored. This study analyzed 18 clinical isolates of A. baumannii from global clones (GC), with draft genomes generated by Illumina sequencing. All isolates were screened for filamentous prophage (FPP) genomes using the Zonula occludens toxin (Zot)-coding gene as a marker. Nine out of the 18 isolates were found to carry zot genes. The complete sequences of four FPPs were predicted. FPPs were exclusively found within GC1, GC7, and GC9 strains. Among the A. baumannii genomes deposited in the NCBI genomic database, FPPs were found to be disseminated in 42 Pasteur STs spanning at least six GCs, most commonly GC1. The impact of FPs on biofilm formation in A. baumannii was investigated using crystal violet assay. None of the zot -negative isolates formed strong biofilms, while six (66.6%) zot -positive isolates did. The biofilm indices of zot -positive isolates were significantly higher compared to zot -negative isolates. The potential enterotoxicity of the zot -positive strains was also assessed using in silico and experimental methods. The cytotoxic effect of cell-free supernatants (CFSs) on Caco-2 cells was measured by the MTT assay. Cells treated with CFSs from zot -positive strains exhibited significantly higher cytotoxicity than those treated with CFSs from zot -negative strains. Upon injecting the CFS of a zot -positive strain intraperitoneally into BALB/c mice, severe diarrhea was observed within 6 h. Histological examination of the intestinal tissue 24 h post-injection revealed significant changes. In conclusion, this study suggests that FPPs are widely disseminated in A. baumannii GCs and may enhance biofilm formation and enterotoxicity, potentially contributing to the pathogen’s virulence.

The deep sea is a massive, largely oligotrophic ecosystem, stretched over nearly 65% of the planet’s surface. Deep-sea planktonic communities are almost completely dependent upon organic carbon sinking from the productive surface, forming a vital component of global biogeochemical cycles. However, despite their importance, viruses from the deep ocean remain largely unknown. Here, we describe the first complete genomes of deep-sea viruses assembled from metagenomic fosmid libraries. “ Candidatus Pelagibacter” (SAR11) phage HTVC010P and Puniceispirillum phage HMO-2011 are considered the most abundant cultured marine viruses known to date. Remarkably, some of the viruses described here recruited as many reads from deep waters as these viruses do in the photic zone, and, considering the gigantic scale of the bathypelagic habitat, these genomes provide information about what could be some of the most abundant viruses in the world at large. Their role in the viral shunt in the global ocean could be very significant. Despite the challenges encountered in inferring the identity of their hosts, we identified one virus predicted to infect members of the globally distributed SAR11 cluster. We also identified a number of putative proviruses from diverse taxa, including deltaproteobacteria, bacteroidetes, SAR11, and gammaproteobacteria. Moreover, our findings also indicate that lysogeny is the preferred mode of existence for deep-sea viruses inhabiting an energy-limited environment, in sharp contrast to the predominantly lytic lifestyle of their photic-zone counterparts. Some of the viruses show a widespread distribution, supporting the tenet “everything is everywhere” for the deep-ocean virome. IMPORTANCE The deep sea is among the largest known habitats and a critical cog in biogeochemical cycling but remains underexplored in its microbiology. Even more than is the case for its prokaryotic community, our knowledge of its viral component has remained limited by the paucity of information provided by studies dependent upon short sequence fragments. In this work, we attempt to fill this existing gap by using a combination of classical fosmid libraries with next-generation sequencing and assembly to recover long viral genomic fragments. We have sequenced ca. 6,000 fosmids from two metagenomics libraries made from prokaryotic biomass from the deep Mediterranean Sea and recovered twenty-eight complete viral genomes, all of them novel and quite distinct from all previously described viral genomes. They are preferentially found in deeper waters and are widely distributed all over the oceans. To our knowledge, this is the first report on complete and cosmopolitan viral genomes from the bathypelagic habitat. The deep sea is among the largest known habitats and a critical cog in biogeochemical cycling but remains underexplored in its microbiology. Even more than is the case for its prokaryotic community, our knowledge of its viral component has remained limited by the paucity of information provided by studies dependent upon short sequence fragments. In this work, we attempt to fill this existing gap by using a combination of classical fosmid libraries with next-generation sequencing and assembly to recover long viral genomic fragments. We have sequenced ca. 6,000 fosmids from two metagenomics libraries made from prokaryotic biomass from the deep Mediterranean Sea and recovered twenty-eight complete viral genomes, all of them novel and quite distinct from all previously described viral genomes. They are preferentially found in deeper waters and are widely distributed all over the oceans. To our knowledge, this is the first report on complete and cosmopolitan viral genomes from the bathypelagic habitat.