Genomic and induction evidence for bacteriophage contributions to sargassum-bacteria symbioses.

BACKGROUND: Symbioses between primary producers and bacteria are crucial for nutrient exchange that fosters host growth and niche adaptation. Yet, how viruses that infect bacteria (phages) influence these bacteria-eukaryote interactions is still largely unknown. Here, we investigate the role of viruses on the genomic diversity and functional adaptations of bacteria associated with pelagic sargassum. This brown alga has dramatically increased its distribution range in the Atlantic in the past decade and is predicted to continue expanding, imposing severe impacts on coastal ecosystems, economies, and human health. RESULTS: We reconstructed 73 bacterial and 3963 viral metagenome-assembled genomes (bMAGs and vMAGs, respectively) from coastal Sargassum natans VIII and surrounding seawater. S. natans VIII bMAGs were enriched in prophages compared to seawater (28% and 0.02%, respectively). Rhodobacterales and Synechococcus bMAGs, abundant members of the S. natans VIII microbiome, were shared between the algae and seawater but were associated with distinct phages in each environment. Genes related to biofilm formation and quorum sensing were enriched in S. natans VIII phages, indicating their potential to influence algal association in their bacterial hosts. In-vitro assays with a bacterial community harvested from sargassum surface biofilms and depleted of free viruses demonstrated that these bacteria are protected from lytic infection by seawater viruses but contain intact and inducible prophages. These bacteria form thicker biofilms when growing on sargassum-supplemented seawater compared to seawater controls, and phage induction using mitomycin C was associated with a significant decrease in biofilm formation. The induced metagenomes were enriched in genomic sequences classified as temperate viruses compared to uninduced controls. CONCLUSIONS: Our data shows that prophages contribute to the flexible genomes of S. natans VIII-associated bacteria. These prophages encode genes with symbiotic functions, and their induction decreases biofilm formation, an essential capacity for flexible symbioses between bacteria and the alga. These results indicate that prophage acquisition and induction contribute to genomic and functional diversification during sargassum-bacteria symbioses, with potential implications for algae growth. Video Abstract.

Novel lytic bacteriophage AhFM11 as an effective therapy against hypervirulent Aeromonas hydrophila.

Several farmed fish species, including carps, tilapia, salmon, and catfish, have experienced significant economic losses in aquaculture due to motile Aeromonas septicemia caused by Aeromonas hydrophila. In the present study, a novel lytic bacteriophage infecting hypervirulent Aeromonas hydrophila (vAh) was isolated and characterized. This is the first report of a phage against vAh. Phage AhFM11 demonstrated lytic activity against both vAh strains and the A. hydrophila reference strain ATCC 35654. The AhFM11 genome was sequenced and assembled, comprising 168,243 bp with an average G + C content of 41.5%. The genome did not harbor any antibiotic resistance genes. Genomic information along with transmission electron microscopy revealed that phage AhFM11 belongs to the Straboviridae family. Therapeutic application of monophage AhFM11 in fish showed 100% survival in injection, 95% in immersion and 93% in oral feeding of phage top-coated feed. Fish and chicken meat spiked with A. hydrophila and phage showed significant reduction of A. hydrophila. These findings support that phage AhFM11 can be used as a biocontrol agent against vAh as an alternative to antibiotics.

A diverse set of Enterococcus-infecting phage provides insight into phage host-range determinants.

Enterococci are robust Gram-positive bacteria that pose a significant threat in healthcare settings due to antibiotic resistance, with vancomycin-resistant enterococci (VRE) most prominent. To tackle this issue, bacteriophages (bacterial viruses) can be exploited as they specifically and efficiently target bacteria. Here, we successfully isolated and characterised a set of novel phages: SHEF10, SHEF11, SHEF13, SHEF14, and SHEF16 which target E. faecalis (SHEF10,11,13), or E. faecium (SHEF13, SHEF14 & SHEF16) strains including a range of clinical and VRE isolates. Genomic analysis shows that all phages are strictly lytic and diverse in terms of genome size and content, quickly and effectively lysing strains at different multiplicity of infections. Detailed analysis of the broad host-range SHEF13 phage revealed the crucial role of the enterococcal polysaccharide antigen (EPA) variable region in its infection of E. faecalis V583. In parallel, the discovery of a carbohydrate-targeting domain (CBM22) found conserved within the three phage genomes indicates a role in cell surface interactions that may be important in phage-bacterial interactons. These findings advance our comprehension of phage-host interactions and pave the way for targeted therapeutic strategies against antibiotic-resistant enterococcal infections.

Bacteriophages isolated from mouse feces attenuates pneumonia mice caused by Pseudomonas aeruginosa.

BACKGROUND: Most of the current bacteriophages (phages) are mostly isolated from environments. However, phages isolated from feces might be more specific to the bacteria that are harmful to the host. Meanwhile, some phages from the environment might affect non-pathogenic bacteria for the host. METHODS: Here, bacteriophages isolated from mouse feces were intratracheally (IT) or intravenously (IV) administered in pneumonia mice caused by Pseudomonas aeruginosa at 2 hours post-intratracheal bacterial administration. As such, the mice with phage treatment, using either IT or IV administration, demonstrated less severe pneumonia as indicated by mortality, serum cytokines, bacteremia, bacterial abundance in bronchoalveolar lavage fluid (BALF), and neutrophil extracellular traps (NETs) in lung tissue (immunofluorescence of neutrophil elastase and myeloperoxidase). RESULTS: Interestingly, the abundance of phages in BALF from the IT and IV injections was similar, supporting a flexible route of phage administration. With the incubation of bacteria with neutrophils, the presence of bacteriophages significantly improved bactericidal activity, but not NETs formation, with the elevated supernatant IL-6 and TNF-α, but not IL-1ß. In conclusion, our findings suggest that bacteriophages against Pseudomonas aeruginosa can be discovered from feces of the host. CONCLUSIONS: The phages attenuate pneumonia partly through an enhanced neutrophil bactericidal activity, but not via inducing NETs formation. The isolation of phages from the infected hosts themselves might be practically useful for future treatment. More studies are warranted.

Soil Giant Phage: Genome and Biological Characteristics of Sinorhizobium Jumbo Phage.

This paper presents the first in-depth research on the biological and genomic properties of lytic rhizobiophage AP-J-162 isolated from the soils of the mountainous region of Dagestan (North Caucasus), which belongs to the centers of origin of cultivated plants, according to Vavilov N.I. The rhizobiophage host strains are nitrogen-fixing bacteria of the genus Sinorhizobium spp., symbionts of leguminous forage grasses. The phage particles have a myovirus virion structure. The genome of rhizobiophage AP-J-162 is double-stranded DNA of 471.5 kb in length; 711 ORFs are annotated and 41 types of tRNAs are detected. The closest phylogenetic relative of phage AP-J-162 is Agrobacterium phage Atu-ph07, but no rhizobiophages are known. The replicative machinery, capsid, and baseplate proteins of phage AP-J-162 are structurally similar to those of Escherichia phage T4, but there is no similarity between their tail protein subunits. Amino acid sequence analysis shows that 339 of the ORFs encode hypothetical or functionally relevant products, while the remaining 304 ORFs are unique. Additionally, 153 ORFs are similar to those of Atu_ph07, with one-third of the ORFs encoding different enzymes. The biological properties and genomic characteristics of phage AP-J-162 distinguish it as a unique model for exploring phage-microbe interactions with nitrogen-fixing symbiotic microorganisms.

Whole-genome sequence of a novel lytic bacteriophage infecting Clavibacter michiganensis subsp. michiganensis from Turkey.

Clavibacter michiganensis subsp. michiganensis (Cmm) is an important plant-pathogenic bacterium that causes canker and wilt diseases. Biological control of the disease with bacteriophages is an alternative to conventional methods. In this study, Phage33 infecting Cmm was characterized based on morphological and genomic properties. Morphological characteristics such as shape and size were investigated using electron microscopy. The whole genome was sequenced using the Illumina Novaseq 6000 platform and the sequence was assembled and annotated. VICTOR and VIRIDIC were used for determining the phylogeny and comparing viral genomes, respectively. Electron microscopy showed that Phage33 has an icosahedral head with a diameter of ~55 nm and a long, thin, non-contractile tail ~169 nm in length. The genome of Phage33 is 56 324 bp in size, has a GC content of 62.49 % and encodes 67 open reading frames. Thirty-seven ORFs showed high homology to functionally annotated bacteriophage proteins in the NCBI database. The remaining 30 ORFs were identified as hypothetical with unknown functions. The genome contains no antimicrobial resistance, no lysogenicity and no virulence signatures, suggesting that it is a suitable candidate for biocontrol agents. The results of a blastn search showed similarity to the previously reported Xylella phage Sano, with an average nucleotide sequence identity of 92.37 % and query coverage of 91 %. This result was verified using VICTOR and VIRIDIC analysis, and suggests that Phage33 is a new member of the genus Sanovirus under the class Caudoviricetes.

Bacteriophage Removal from Infected Salmonella Cultures.

Bacteriophages, or simply phages, play a vital role in microbial environments, impacting bacterial populations and shaping their evolution and interactions. These organisms are viruses that infect and replicate within bacterial hosts. Phages are ubiquitous on Earth, highly diverse, and very abundant. While bacteriophages have valuable roles in different environments and are a key area of research in microbiology and ecology, their presence can be undesirable in certain industrial processes or products. Considering the abundance and ubiquity of bacteriophages on Earth, the design of procedures for the removal of bacteriophages from bacterial cultures is crucial in diverse laboratory and industrial applications to preserve the integrity of the cultures and ensure accurate experimental results or product quality. Here, we have fine-tuned a protocol to eliminate the bacteriophages from infected Salmonella enterica cultures, using a strategy based on the use of lipopolysaccharides (LPS) located in the outer membrane of Gram-negative bacteria. Bacterial LPS plays an important role in host recognition by phages, and we make use of this property to design an effective procedure for the removal of phages, which use LPS as a receptor, in Salmonella bacterial cultures.

fENko-Kae01 is a flagellum-specific jumbo phage infecting Klebsiella aerogenes.

BACKGROUND: Klebsiella aerogenes is an opportunistic pathogen that causes a wide variety of infections. Due to the rising problem of antibiotic resistance, novel antibiotics and strategies to combat bacterial infections are needed. Host-specific bacteriophages are natural enemies of bacteria and can be used in phage therapy as an alternative form of treatment against bacterial infections. Jumbo phages are defined as phages with genomes larger than 200 kb. Relatively few studies have been done on jumbo phages compared to smaller phages. RESULTS: A novel phage, fENko-Kae01, was isolated from a commercial phage cocktail. Genomic analysis revealed that fENko-Kae01 is a lytic jumbo phage with a 360 kb genome encoding 578 predicted genes. No highly similar phage genomes were identified and fENko-Kae01 may be a completely new genus representative. No known genes associated with lysogenic life cycle, bacterial virulence, or antibiotic resistance were identified. The phage had myovirus morphology and a narrow host range. Phage resistant bacterial mutants emerged under phage selection. Whole genome sequencing revealed that the biogenesis of the flagellum was affected in four mutants and the lack of functional flagellum was confirmed in motility assays. Furthermore, phage fENKo-Kae01 failed to adsorb on the non-motile mutants indicating that the bacterial flagellum is the phage-binding receptor. CONCLUSIONS: fENko-Kae01 is a novel jumbo bacteriophage that is considered safe for phage therapy. fENko-Kae01 uses the flagellum as the phage-binding receptor and may represent a completely novel genus.

Isolation and characterization of bacteriophages against E.coli urinary tract infection and evaluating their anti-biofilm activity and antibiotic synergy.

Urinary tract infections (UTIs) by Uropathogenic Escherichia coli (UPEC) are a significant health concern, especially due to the increasing prevalence of antibiotic resistance. This study focuses on isolating and characterizing bacteriophages specific to UPEC strains isolated from UTI samples. The isolated phages were assessed for their ability to target and lyse UPEC in vitro, focusing on their efficacy in disrupting biofilms, a key virulence factor contributing to UTI recurrence and antibiotic resistance. The morphological structure observed by TEM belongs to Myoviridae, the phage exhibited icosahedral symmetry with a long non-constricting tail, the approximate measurement of the phage head was 39 nm in diameter, and the phage tail was 105.317 nm in length. One-step growth experiments showed that the latent period was approximately 20 min, followed by a rise period of 40 min, and a growth plateau was reached within 20 min and the burst size observed was 26 phages/infected bacterial cells. These phages were capable of killing cells within the biofilms, leading to a reduction in living cell counts after a single treatment. This study highlights the potential of phages to play a significant role in disrupting, inactivating, and destroying Uropathogenic Escherichia coli (UPEC) biofilms. Such findings could be instrumental in developing treatment strategies that complement antibiotics and disinfectants. The phage-antibiotic synergistic activity was compared to have the possibility to facilitate the advancement of focused and enduring alternatives to traditional antibiotic therapies for UTIs.

Assessment of crAssphage as a biological variable for SARS-CoV-2 data normalization in wastewater surveillance.

AIMS: This study aimed to assess the use of cross-assembled phage (crAssphage) as an endogenous control employing a multivariate normalization analysis and its application as a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) data normalizer. METHODS AND RESULTS: A total of 188 twelve-hour composite raw sewage samples were obtained from eight wastewater treatment plants (WWTP) during a 1-year monitoring period. Employing the N1 and N2 target regions, SARS-CoV-2 RNA was detected in 94% (177) and 90% (170) of the samples, respectively, with a global median of 5 log10 genomic copies per liter (GC l-1). CrAssphage was detected in 100% of the samples, ranging from 8.29 to 10.43 log10 GC l-1, with a median of 9.46 ± 0.40 log10 GC l-1, presenting both spatial and temporal variabilities. CONCLUSIONS: Although SARS-CoV-2 data normalization employing crAssphage revealed a correlation with clinical cases occurring during the study period, crAssphage normalization by the flow per capita per day of each WWTP increased this correlation, corroborating the importance of normalizing wastewater surveillance data in disease trend monitoring.

Overcoming donor variability and risks associated with fecal microbiota transplants through bacteriophage-mediated treatments.

BACKGROUND: Fecal microbiota transplantation (FMT) and fecal virome transplantation (FVT, sterile filtrated donor feces) have been effective in treating recurrent Clostridioides difficile infections, possibly through bacteriophage-mediated modulation of the gut microbiome. However, challenges like donor variability, costly screening, coupled with concerns over pathogen transfer (incl. eukaryotic viruses) with FMT or FVT hinder their wider clinical application in treating less acute diseases. METHODS: To overcome these challenges, we developed methods to broaden FVT's clinical application while maintaining efficacy and increasing safety. Specifically, we employed the following approaches: (1) chemostat-fermentation to reproduce the bacteriophage FVT donor component and remove eukaryotic viruses (FVT-ChP), (2) solvent-detergent treatment to inactivate enveloped viruses (FVT-SDT), and (3) pyronin-Y treatment to inhibit RNA virus replication (FVT-PyT). We assessed the efficacy of these processed FVTs in a C. difficile infection mouse model and compared them with untreated FVT (FVT-UnT), FMT, and saline. RESULTS: FVT-SDT, FVT-UnT, and FVT-ChP reduced the incidence of mice reaching the humane endpoint (0/8, 2/7, and 3/8, respectively) compared to FMT, FVT-PyT, and saline (5/8, 7/8, and 5/7, respectively) and significantly reduced the load of colonizing C. difficile cells and associated toxin A/B levels. There was a potential elimination of C. difficile colonization, with seven out of eight mice treated with FVT-SDT testing negative with qPCR. In contrast, all other treatments exhibited the continued presence of C. difficile. Moreover, the results were supported by changes in the gut microbiome profiles, cecal cytokine levels, and histopathological findings. Assessment of viral engraftment following FMT/FVT treatment and host-phage correlations analysis suggested that transfer of phages likely were an important contributing factor associated with treatment efficacy. CONCLUSIONS: This proof-of-concept study shows that specific modifications of FVT hold promise in addressing challenges related to donor variability and infection risks. Two strategies lead to treatments significantly limiting C. difficile colonization in mice, with solvent/detergent treatment and chemostat propagation of donor phages emerging as promising approaches. Video Abstract.

Three novel Enterobacter cloacae bacteriophages for therapeutic use from Ghanaian natural waters.

Infections caused by multidrug-resistant (MDR) bacteria are a growing global concern. Enterobacter cloacae complex (ECC) species are particularly adept at developing antibiotic resistance. Phage therapy is proposed as an alternative treatment for pathogens that no longer respond to antibiotics. Unfortunately, ECC phages are understudied when compared to phages of many other bacterial species. In this Ghanaian-Finnish study, we isolated two ECC strains from ready-to-eat food samples and three novel phages from natural waters against these strains. We sequenced the genomic DNA of the novel Enterobacter phages, fGh-Ecl01, fGh-Ecl02, and fGh-Ecl04, and assessed their therapeutic potential. All of the phages were found to be lytic, easy to propagate, and lacking any toxic, integrase, or antibiotic resistance genes and were thus considered suitable for therapy purposes. They all were found to be related to T4-type viruses: fGh-Ecl01 and fGh-Ecl04 to karamviruses and fGh-Ecl02 to agtreviruses. Testing of Finnish clinical ECC strains showed promising susceptibility to these novel phages. As many as 61.1% of the strains were susceptible to fGh-Ecl01 and fGh-Ecl04, and 7.4% were susceptible to fGh-Ecl02. Finally, we investigated the susceptibility of the newly isolated ECC strains to three antibiotics - meropenem, ciprofloxacin, and cefepime - in combination with the novel phages. The use of phages and antibiotics together had synergistic effects. When using an antibiotic-phage combination, even low concentrations of antibiotics fully inhibited the growth of bacteria.

Distributions, interactions, and dynamics of prokaryotes and phages in a hybrid biological wastewater treatment system.

BACKGROUND: Understanding the interactions and dynamics of microbiotas within biological wastewater treatment systems is essential for ensuring their stability and long-term sustainability. In this study, we developed a systematic framework employing multi-omics and Hi-C sequencing to extensively investigate prokaryotic and phage communities within a hybrid biofilm and activated sludge system. RESULTS: We uncovered distinct distribution patterns, metabolic capabilities, and activities of functional prokaryotes through the analysis of 454 reconstructed prokaryotic genomes. Additionally, we reconstructed a phage catalog comprising 18,645 viral operational taxonomic units (vOTUs) with high length and contiguity using hybrid assembly, and a distinct distribution of phages was depicted between activated sludge (AS) and biofilm. Importantly, 1340 host-phage pairs were established using Hi-C and conventional in silico methods, unveiling the host-determined phage prevalence. The majority of predicted hosts were found to be involved in various crucial metabolic processes, highlighting the potential vital roles of phages in influencing substance metabolism within this system. Moreover, auxiliary metabolic genes (AMGs) related to various categories (e.g., carbohydrate degradation, sulfur metabolism, transporter) were predicted. Subsequent activity analysis emphasized their potential ability to mediate host metabolism during infection. We also profiled the temporal dynamics of phages and their associated hosts using 13-month time-series metagenomic data, further demonstrating their tight interactions. Notably, we observed lineage-specific infection patterns, such as potentially host abundance- or phage/host ratio-driven phage population changes. CONCLUSIONS: The insights gained from this research contribute to the growing body of knowledge surrounding interactions and dynamics of host-phage and pave the way for further exploration and potential applications in the field of microbial ecology. Video Abstract.

Isolation of lytic bacteriophages and their relationships with the adherence genes of Staphylococcus saprophyticus.

OBJECTIVE: This study aimed to introduce a lytic bacteriophage against Staphylococcus saprophyticus from wastewater in Gorgan, northern Iran. RESULTS: The vB_SsapS-46 phage was isolated from urban wastewater and formed round and clear plaques on bacterial culture. It was visualized by electron microscopy and had a large head (approximately 106 nm) and a long tail (approximately 150 nm), indicating that it belongs to the Siphoviridae family. The host range of vB_SsapS-46 was determined using a spot test on 35 S. saprophyticus clinical isolates, and it was able to lyse 12 of the 35 clinical isolates (34%). Finally, the relationship between phage sensitivity and adherence genes was assessed, revealing no significant correlation between phage sensitivity and the frequency of adherence genes. The vB_SsapS-46 phage can be used alone or in a mixture in future studies to control urinary tract infections caused by this bacterium, especially in the elimination of drug-resistant pathogens.

A novel flavobacterial phage abundant during green tide, representing a new viral family, Zblingviridae.

Flavobacteriia are the dominant and active bacteria during algal blooms and play an important role in polysaccharide degradation. However, little is known about phages infecting Flavobacteriia, especially during green tide. In this study, a novel virus, vB_TgeS_JQ, infecting Flavobacteriia was isolated from the surface water of the Golden Beach of Qingdao, China. Transmission electron microscopy demonstrated that vB_TgeS_JQ had the morphology of siphovirus. The experiments showed that it was stable from -20°C to 45°C and pH 5 to pH 8, with latent and burst periods both lasting for 20 min. Genomic analysis showed that the phage vB_TgeS_JQ contained a 40,712-bp dsDNA genome with a GC content of 30.70%, encoding 74 open-reading frames. Four putative auxiliary metabolic genes were identified, encoding electron transfer-flavoprotein dehydrogenase, calcineurin-like phosphoesterase, phosphoribosyl-ATP pyrophosphohydrolase, and TOPRIM nucleotidyl hydrolase. The abundance of phage vB_TgeS_JQ was higher during Ulva prolifera (U. prolifera) blooms compared with other marine environments. The phylogenetic and comparative genomic analyses revealed that vB_TgeS_JQ exhibited significant differences from all other phage isolates in the databases and therefore was classified as an undiscovered viral family, named Zblingviridae. In summary, this study expands the knowledge about the genomic, phylogenetic diversity and distribution of flavobacterial phages (flavophages), especially their roles during U. prolifera blooms. IMPORTANCE: The phage vB_TgeS_JQ was the first flavobacterial phage isolated during green tide, representing a new family in Caudoviricetes and named Zblingviridae. The abundance of phage vB_TgeS_JQ was higher during the Ulva prolifera blooms. This study provides insights into the genomic, phylogenetic diversity, and distribution of flavophages, especially their roles during U. prolifera blooms.

Viromic and Metagenomic Analyses of Commercial Spirulina Fermentations Reveal Remarkable Microbial Diversity.

Commercially produced cyanobacteria preparations sold under the name spirulina are widely consumed, due to their traditional use as a nutrient-rich foodstuff and subsequent marketing as a superfood. Despite their popularity, the microbial composition of ponds used to cultivate these bacteria is understudied. A total of 19 pond samples were obtained from small-scale spirulina farms and subjected to metagenome and/or virome sequencing, and the results were analysed. A remarkable level of prokaryotic and viral diversity was found to be present in the ponds, with Limnospira sp. and Arthrospira sp. sometimes being notably scarce. A detailed breakdown of prokaryotic and viral components of 15 samples is presented. Twenty putative Limnospira sp.-infecting bacteriophage contigs were identified, though no correlation between the performance of these cultures and the presence of phages was found. The high diversity of these samples prevented the identification of clear trends in sample performance over time, between ponds or when comparing successful and failed fermentations.

Fecal microbiota transplantation alters gut phage communities in a clinical trial for obesity.

BACKGROUND: Fecal microbiota transplantation (FMT) is a therapeutic intervention used to treat diseases associated with the gut microbiome. In the human gut microbiome, phages have been implicated in influencing human health, with successful engraftment of donor phages correlated with FMT treatment efficacy. The impact that gastrointestinal phages exert on human health has primarily been connected to their ability to modulate the bacterial communities in the gut. Nonetheless, how FMT affects recipients' phage populations, and in turn, how this influences the gut environment, is not yet fully understood. In this study, we investigated the effects of FMT on the phageome composition of participants within the Gut Bugs Trial (GBT), a double-blind, randomized, placebo-controlled trial that investigated the efficacy of FMT in treating obesity and comorbidities in adolescents. Stool samples collected from donors at the time of treatment and recipients at four time points (i.e., baseline and 6 weeks, 12 weeks, and 26 weeks post-intervention), underwent shotgun metagenomic sequencing. Phage sequences were identified and characterized in silico to examine evidence of phage engraftment and to assess the extent of FMT-induced alterations in the recipients' phageome composition. RESULTS: Donor phages engrafted stably in recipients following FMT, composing a significant proportion of their phageome for the entire course of the study (33.8 ± 1.2% in females and 33.9 ± 3.7% in males). Phage engraftment varied between donors and donor engraftment efficacy was positively correlated with their phageome alpha diversity. FMT caused a shift in recipients' phageome toward the donors' composition and increased phageome alpha diversity and variability over time. CONCLUSIONS: FMT significantly altered recipients' phage and, overall, microbial populations. The increase in microbial diversity and variability is consistent with a shift in microbial population dynamics. This proposes that phages play a critical role in modulating the gut environment and suggests novel approaches to understanding the efficacy of FMT in altering the recipient's microbiome. TRIAL REGISTRATION: The Gut Bugs Trial was registered with the Australian New Zealand Clinical Trials Registry (ACTR N12615001351505). Trial protocol: the trial protocol is available at https://bmjopen.bmj.com/content/9/4/e026174 . Video Abstract.

Large-scale single-virus genomics uncovers hidden diversity of river water viruses and diversified gene profiles.

Environmental viruses (primarily bacteriophages) are widely recognized as playing an important role in ecosystem homeostasis through the infection of host cells. However, the majority of environmental viruses are still unknown as their mosaic structure and frequent mutations in their sequences hinder genome construction in current metagenomics. To enable the large-scale acquisition of environmental viral genomes, we developed a new single-viral genome sequencing platform with microfluidic-generated gel beads. Amplification of individual DNA viral genomes in mass-produced gel beads allows high-throughput genome sequencing compared to conventional single-virus genomics. The sequencing analysis of river water samples yielded 1431 diverse viral single-amplified genomes, whereas viral metagenomics recovered 100 viral metagenome-assembled genomes at the comparable sequence depth. The 99.5% of viral single-amplified genomes were determined novel at the species level, most of which could not be recovered by a metagenomic assembly. The large-scale acquisition of diverse viral genomes identified protein clusters commonly detected in different viral strains, allowing the gene transfer to be tracked. Moreover, comparative genomics within the same viral species revealed that the profiles of various methyltransferase subtypes were diverse, suggesting an enhanced escape from host bacterial internal defense mechanisms. Our use of gel bead-based single-virus genomics will contribute to exploring the nature of viruses by accelerating the accumulation of draft genomes of environmental DNA viruses.

A novel Saclayvirus Acinetobacter baumannii phage genomic analysis and effectiveness in preventing pneumonia.

Acinetobacter baumannii, which is resistant to multiple drugs, is an opportunistic pathogen responsible for severe nosocomial infections. With no antibiotics available, phages have obtained clinical attention. However, since immunocompromised patients are often susceptible to infection, the appropriate timing of administration is particularly important. During this research, we obtained a lytic phage vB_AbaM_P1 that specifically targets A. baumannii. We then assessed its potential as a prophylactic treatment for lung infections caused by clinical strains. The virus experiences a period of inactivity lasting 30 min and produces approximately 788 particles during an outbreak. Transmission electron microscopy shows that vB_AbaM_P1 was similar to the Saclayvirus. Based on the analysis of high-throughput sequencing and bioinformatics, vB_AbaM_P1 consists of 107537 bases with a G + C content of 37.68%. It contains a total of 177 open reading frames and 14 tRNAs. No antibiotic genes were detected. In vivo experiments, using a cyclophosphamide-induced neutrophil deficiency model, tested the protective effect of phage on neutrophil-deficient rats by prophylactic application of phage. The use of phages resulted in a decrease in rat mortality caused by A. baumannii and a reduction in the bacterial burden in the lungs. Histologic examination of lung tissue revealed a decrease in the presence of immune cells. The presence of phage vB_AbaM_P1 had a notable impact on preventing A. baumannii infection, as evidenced by the decrease in oxidative stress in lung tissue and cytokine levels in serum. Our research offers more robust evidence for the early utilization of bacteriophages to mitigate A. baumannii infection. KEY POINTS: •A novel Saclayvirus phage infecting A. baumannii was isolated from sewage. •The whole genome was determined, analyzed, and compared to other phages. •Assaying the effect of phage in preventing infection in neutrophil-deficient models.

Gut virome-wide association analysis identifies cross-population viral signatures for inflammatory bowel disease.

BACKGROUND: The gut virome has been implicated in inflammatory bowel disease (IBD), yet a full understanding of the gut virome in IBD patients, especially across diverse geographic populations, is lacking. RESULTS: In this study, we conducted a comprehensive gut virome-wide association study in a Chinese cohort of 71 IBD patients (15 with Crohn's disease and 56 with ulcerative colitis) and 77 healthy controls via viral-like particle (VLP) and bulk virome sequencing of their feces. By utilizing an integrated gut virus catalog tailored to the IBD virome, we revealed fundamental alterations in the gut virome in IBD patients. These characterized 139 differentially abundant viral signatures, including elevated phages predicted to infect Escherichia, Klebsiella, Enterococcus_B, Streptococcus, and Veillonella species, as well as IBD-depleted phages targeting Prevotella, Ruminococcus_E, Bifidobacterium, and Blautia species. Remarkably, these viral signatures demonstrated high consistency across diverse populations such as those in Europe and the USA, emphasizing their significance and broad relevance in the disease context. Furthermore, fecal virome transplantation experiments verified that the colonization of these IBD-characterized viruses can modulate experimental colitis in mouse models. CONCLUSIONS: Building upon these insights into the IBD gut virome, we identified potential biomarkers for prognosis and therapy in IBD patients, laying the foundation for further exploration of viromes in related conditions. Video Abstract.