Phages are pivotal in shaping microbial communities and biogeochemical cycles, while our understanding of the diversity, functions potential, and resistance gene carriage of phages in hospital wastewater (HWW) remains limited. We collected influent and effluent samples from the 3 hospital wastewater treatment plants (HWTPs) to assess the diversity and fate of phages, the interactions between phages and hosts, and the presence of resistance genes and auxiliary metabolic genes (AMGs) encoded by phages. Compared to influent, effluent showed reduced phage abundance and altered composition, with decreases in Microviridae and Inoviridae. The gene-sharing network highlights that many phages in HWW are not classified in known viral genera, suggesting HWW as a rich source of new viruses. There was a significant association between phages and microorganisms, with approximately 32.57 % of phages expected to be capable of infecting microbial hosts, characterized primarily by lytic activity. A total of 8 unique antibiotic resistance genes, 13 unique metal resistance genes, and 5 mobile genetic elements were detected in 3 HWTPs phageomes. Phage AMGs have the potential to influence carbon, nitrogen, phosphorus, and sulfur metabolism, impacting biogeochemical cycles. This study reveals the genomic diversity and ecological role of phages in HWTPs, highlighting their environmental and ecosystem impact.

The bacterial diseases black leg and soft rot in potatoes cause heavy losses of potatoes worldwide. Bacteria within the genus Pectobacteriaceae are the causative agents of black leg and soft rot. The use of antibiotics in agriculture is heavily regulated and no other effective treatment currently exists, but bacteriophages (phages) have shown promise as potential biocontrol agents. In this study we isolated soft rot bacteria from potato tubers and plant tissue displaying soft rot or black leg symptoms collected in Danish fields. We then used the isolated bacterial strains as hosts for phage isolation. Using organic waste, we isolated phages targeting different species within Pectobacterium. Here we focus on seven of these phages representing a new genus primarily targeting P. brasiliense; phage Ymer, Amona, Sabo, Abuela, Koroua, Taid and Pappous. TEM image of phage Ymer showed siphovirus morphotype, and the proposed Ymer genus belongs to the class Caudoviricetes, with double-stranded DNA genomes varying from 39 kb to 43 kb. In silico host range prediction using a CRISPR-Cas spacer database suggested both P. brasiliense, P. polaris and P. versatile as natural hosts for phages within the proposed Ymer genus. A following host range experiment, using 47 bacterial isolates from Danish tubers and plants symptomatic with soft rot or black leg disease verified the in silico host range prediction, as the genus as a group were able to infect all three Pectobacterium species. Phages did, however, primarily target P. brasiliense isolates and displayed differences in host range even within the species level. Two of the phages were able to infect two or more Pectobacterium species. Despite no nucleotide similarity with any phages in the NCBI database, the proposed Ymer genus did share some similarity at the protein level, as well as gene synteny, with currently known phages. None of the phages encoded integrases or other genes typically associated with lysogeny. Similarly, no virulence factors nor antimicrobial resistance genes were found, and combined with their ability to infect several soft rot-causing Pectobacterium species from Danish fields, demonstrates their potential as biocontrol agents against soft rot and black leg diseases in potatoes.

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In this study, we have investigated innate immune activation capacity and metabolic features of a population of P. aeruginosa PAO1 phage-resistant mutants with diverse genetic modification (large genomic deletions and point mutations) arising after exposure to phages targetting lipopolysaccharide (LPS) or Type-4 pili (T4P). Deletions led to the loss of genes involved in LPS synthesis, cell envelope permeability, efflux systems, biofilm production, oxidative stress tolerance, and DNA repair. Loss of LPS O antigen resulted in bacterial sensitivity to serum complement and stimulation of inflammatory cascades but did not cause increased phagocytosis, while T4P phage-resistant mutants were more effectively phagocytized than LPS-defective mutants. Changes in the utilization of different carbon, nitrogen, sulphur, and phosphorus sources were identified, especially in mutants where the two phage DNA persisted in the bacterial population (pseudolysogeny). However, the metabolic changes did not directly correlate with single-gene mutations or the large gene deletions, suggesting they reflect adaptive changes to the gene modifications that arise during the selection of resistant mutants. In contrast, phage-resistant mutants were susceptible to humoral innate immune responses, suggesting that phage resistance may be a beneficial outcome of phage therapy.

BACKGROUND: Viruses play important roles in modulating microbial communities and influencing global biogeochemistry. There is now growing interest in characterising their ecological roles across diverse biomes. However, little is known about viral ecology in low-nutrient, chemotrophic-based environments. In such ecosystems, virus-driven manipulation of nutrient cycles might have profound impacts across trophic levels. In particular, anchialine environments, which are low-energy underground estuaries sustained by chemotrophic processes, represent ideal model systems to study novel virus-host-environment interactions.
RESULTS: Here, we employ metagenomic sequencing to investigate the viral community in Bundera Sinkhole, an anchialine ecosystem rich in endemic species supported by microbial chemosynthesis. We find that the viruses are highly novel, with less than 2% representing described viruses, and are hugely abundant, making up as much as 12% of microbial intracellular DNA. These highly abundant viruses largely infect important prokaryotic taxa that drive key metabolic processes in the sinkhole. Further, the abundance of viral auxiliary metabolic genes (AMGs) involved in nucleotide and protein synthesis was strongly correlated with declines in environmental phosphate and sulphate concentrations. These AMGs encoded key enzymes needed to produce sulphur-containing amino acids, and phosphorus metabolic enzymes involved in purine and pyrimidine nucleotide synthesis. We hypothesise that this correlation is either due to selection of these AMGs under low phosphate and sulphate concentrations, highlighting the dynamic interactions between viruses, their hosts, and the environment; or, that these AMGs are driving increased viral nucleotide and protein synthesis via manipulation of host phosphorus and sulphur metabolism, consequently driving nutrient depletion in the surrounding water.
CONCLUSIONS: This study represents the first metagenomic investigation of viruses in anchialine ecosystems, and provides new hypotheses and insights into virus-host-environment interactions in such \'dark\', low-energy environments. This is particularly important since anchialine ecosystems are characterised by diverse endemic species, both in their microbial and faunal assemblages, which are primarily supported by microbial chemosynthesis. Thus, virus-host-environment interactions could have profound effects cascading through all trophic levels.

Emerging evidence suggests that due to the misuse of antibiotics, bacteriophage (phage) therapy has been recognized as one of the most promising strategies for treating human diseases infected by antibiotic-resistant bacteria. Identification of phage-host interactions (PHIs) can help to explore the mechanisms of bacterial response to phages and provide new insights into effective therapeutic approaches. Compared to conventional wet-lab experiments, computational models for predicting PHIs can not only save time and cost, but also be more efficient and economical. In this study, we developed a deep learning predictive framework called GSPHI to identify potential phage and target bacterium pairs through DNA and protein sequence information. More specifically, GSPHI first initialized the node representations of phages and target bacterial hosts via a natural language processing algorithm. Then a graph embedding algorithm structural deep network embedding (SDNE) was utilized to extract local and global information from the interaction network, and finally, a deep neural network (DNN) was applied to accurately detect the interactions between phages and their bacterial hosts. In the drug-resistant bacteria dataset ESKAPE, GSPHI achieved a prediction accuracy of 86.65 % and AUC of 0.9208 under the 5-fold cross-validation technique, significantly better than other methods. In addition, case studies in Gram-positive and negative bacterial species demonstrated that GSPHI is competent in detecting potential Phage-host interactions. Taken together, these results indicate that GSPHI can provide reasonable candidate sensitive bacteria to phages for biological experiments. The webserver of the GSPHI predictor is freely available at http://120.77.11.78/GSPHI/.

Phages and their bacterial hosts together constitute a vast and diverse ecosystem. Facing the infection of phages, prokaryotes have evolved a wide range of antiviral mechanisms, and phages in turn have adopted multiple tactics to circumvent or subvert these mechanisms to survive. An in-depth investigation into the interaction between phages and bacteria not only provides new insight into the ancient coevolutionary conflict between them but also produces precision biotechnological tools based on anti-phage systems. Moreover, a more complete understanding of their interaction is also critical for the phage-based antibacterial measures. Compared to the bacterial antiviral mechanisms, studies into counter-defense strategies adopted by phages have been a little slow, but have also achieved important advances in recent years. In this review, we highlight the numerous intracellular immune systems of bacteria as well as the countermeasures employed by phages, with an emphasis on the bacteriophage strategies in response to host antiviral immunity.

Filamentous phages are ubiquitously distributed in the global oceans. However, little is known about their biological contribution to their host\'s genetic and phenotypic diversity. In this study, a filamentous phage, Vaf1, was isolated and characterized from the emerging marine pathogen strain Vibrio alginolyticus AP-1. We explored the effects of the resident phage Vaf1 on the host physiology under diverse conditions by precisely deleting the entire phage Vaf1. Our results demonstrate that the presence of phage Vaf1 significantly increased biofilm formation, swarming motility, and contact-dependent competition. Furthermore, the gene expression profile suggests that several phage genes were upregulated in response to low-nutrient conditions. Unexpectedly, an in vivo study of zebrafish shows that fish infected with strain ΔVaf1 survived longer than those infected with wild-type strain AP-1, indicating that Vaf1 contributes to the virulence of V. alginolyticus. Together, our results provide direct evidence for the effect of Vaf1 phage-mediated phenotypic changes in marine bacteria V. alginolyticus. This further emphasizes the impressive complexity and diversity that filamentous phage-host interactions pose and the challenges associated with bacterial disease control in marine aquaculture. IMPORTANCE Non-lytic filamentous phages can replicate without killing their host, establishing long-term persistence within the bacterial host. In contrast to the well-studied CTXφ phage of the human-pathogenic Vibrio cholerae, little is known about the filamentous phage Vaf1 and its biological role in host fitness. In this study, we constructed a filamentous phage-deleted strain, ΔVaf1, and provided direct evidence on how an intact phage, φVaf1, belonging to the family Inoviridae, helps the bacterial host AP-1 to overcome adverse environmental conditions. Our results likely open new avenues for fundamental studies on how filamentous phage-host interactions regulate different aspects of Vibrio cell behaviors.

Bacteriophages in the family Inoviridae, or inoviruses, are under-characterized phages previously implicated in bacterial pathogenesis by contributing to biofilm formation, immune evasion, and toxin secretion. Unlike most bacteriophages, inoviruses do not lyse their host cells to release new progeny virions; rather, they encode a secretion system that actively pumps them out of the bacterial cell. To date, no inovirus associated with the human gut microbiome has been isolated or characterized.
In this study, we utilized in silico, in vitro, and in vivo methods to detect inoviruses in bacterial members of the gut microbiota. By screening a representative genome library of gut commensals, we detected inovirus prophages in Enterocloster spp. (formerly Clostridium spp.). We confirmed the secretion of inovirus particles in in vitro cultures of these organisms using imaging and qPCR. To assess how the gut abiotic environment, bacterial physiology, and inovirus secretion may be linked, we deployed a tripartite in vitro assay that progressively evaluated bacterial growth dynamics, biofilm formation, and inovirus secretion in the presence of changing osmotic environments. Counter to other inovirus-producing bacteria, inovirus production was not correlated with biofilm formation in Enterocloster spp. Instead, the Enterocloster strains had heterogeneous responses to changing osmolality levels relevant to gut physiology. Notably, increasing osmolality induced inovirus secretion in a strain-dependent manner. We confirmed inovirus secretion in a gnotobiotic mouse model inoculated with individual Enterocloster strains in vivo in unperturbed conditions. Furthermore, consistent with our in vitro observations, inovirus secretion was regulated by a changed osmotic environment in the gut due to osmotic laxatives.
In this study, we report on the detection and characterization of novel inoviruses from gut commensals in the Enterocloster genus. Together, our results demonstrate that human gut-associated bacteria can secrete inoviruses and begin to elucidate the environmental niche filled by inoviruses in commensal bacteria. Video Abstract.

Therapeutic bacteriophages (phages) are being considered as alternatives in the fight against Clostridioides difficile infections. To be efficient, phages should have a wide host range, buthe lack of knowledge about the cell receptor used by C. difficile phages hampers the rational design of phage cocktails. Recent reports suggested that the C. difficile surface layer protein A (SlpA) is an important phage receptor, but available data are still limited. Here, using the epidemic R20291 strain and its FM2.5 mutant derivative lacking a functional S-layer, we show that the absence of SlpA renders cells completely resistant to infection by ϕCD38-2, ϕCD111, and ϕCD146, which normally infect the parental strain. Complementation with 12 different S-layer cassette types (SLCTs) expressed from a plasmid revealed that SLCT-6 also allowed infection by ϕCD111 and SLCT-11 enabled infection by ϕCD38-2 and ϕCD146. Of note, the expression of SLCT-1, -6, -8, -9, -10, or -12 conferred susceptibility to infection by 5 myophages that normally do not infect the R20291 strain. Also, deletion of the D2 domain within the low-molecular-weight fragment of SlpA was found to abolish infection by ϕCD38-2 and ϕCD146 but not ϕCD111. Altogether, our data suggest that many phages use SlpA as their receptor and, most importantly, that both siphophages and myophages target SlpA despite major differences in their tail structures. Our study therefore represents an important step in understanding the interactions between C. difficile and its phages. IMPORTANCE Phage therapy represents an interesting alternative to treat Clostridioides difficile infections because, contrary to antibiotics, most phages are highly species specific, thereby sparing the beneficial gut microbes that protect from infection. However, currently available phages against C. difficile have a narrow host range and target members from only one or a few PCR ribotypes. Without a clear comprehension of the factors that define host specificity, and in particular the host receptor recognized by phages, it is hard to develop therapeutic cocktails in a rational manner. In our study, we provide clear and unambiguous experimental evidence that SlpA is a common receptor used by many siphophages and myophages. Although work is still needed to define how a particular phage receptor-binding protein binds to a specific SLCT, the identification of SlpA as a common receptor is a major keystone that will facilitate the rational design of therapeutic phage cocktails against clinically important strains.