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Viral sewage metagenomics is a novel field of study used for surveillance, epidemiological studies, and evaluation of waste water treatment efficiency. In raw sewage human waste is mixed with household, industrial and drainage water, and virus particles are, therefore, only found in low concentrations. This necessitates a step of sample concentration to allow for sensitive virus detection. Additionally, viruses harbor a large diversity of both surface and genome structures, which makes universal viral genomic extraction difficult. Current studies have tackled these challenges in many different ways employing a wide range of viral concentration and extraction procedures. However, there is limited knowledge of the efficacy and inherent biases associated with these methods in respect to viral sewage metagenomics, hampering the development of this field. By the use of next generation sequencing this study aimed to evaluate the efficiency of four commonly applied viral concentrations techniques (precipitation with polyethylene glycol, organic flocculation with skim milk, monolithic adsorption filtration and glass wool filtration) and extraction methods (Nucleospin RNA XS, QIAamp Viral RNA Mini Kit, NucliSENS® miniMAG®, or PowerViral® Environmental RNA/DNA Isolation Kit) to determine the viriome in a sewage sample. We found a significant influence of concentration and extraction protocols on the detected viriome. The viral richness was largest in samples extracted with QIAamp Viral RNA Mini Kit or PowerViral® Environmental RNA/DNA Isolation Kit. Highest viral specificity were found in samples concentrated by precipitation with polyethylene glycol or extracted with Nucleospin RNA XS. Detection of viral pathogens depended on the method used. These results contribute to the understanding of method associated biases, within the field of viral sewage metagenomics, making evaluation of the current literature easier and helping with the design of future studies.

Phages are viruses of bacteria and are the smallest and most common biological entities in the environment. They can reproduce immediately after infection or integrate as a prophage into their host genome. SPβ is a prophage of the Gram-positive model organism Bacillus subtilis 168, and it has been known for more than 50 years. It is sensitive to dsDNA damage and is induced through exposure to mitomycin C or UV radiation. When induced from the prophage, SPβ requires 90 min to produce and release about 30 virions. Genomes of sequenced related strains range between 128 and 140 kb, and particle-packed dsDNA exhibits terminal redundancy. Formed particles are of the Siphoviridae morphotype. Related isolates are known to infect other B.subtilis clade members. When infecting a new host, SPβ presumably follows a two-step strategy, adsorbing primarily to teichoic acid and secondarily to a yet unknown factor. Once in the host, SPβ-related phages pass through complex lysis–lysogeny decisions and either enter a lytic cycle or integrate as a dormant prophage. As prophages, SPβ-related phages integrate at the host chromosome's replication terminus, and frequently into the spsM or kamA gene. As a prophage, it imparts additional properties to its host via phage-encoded proteins. The most notable of these functional proteins is sublancin 168, which is used as a molecular weapon by the host and ensures prophage maintenance. In this review, we summarise the existing knowledge about the biology of the phage regarding its life cycle and discuss its potential as a research object.

Salmonella enteritidis is one of the most common pathogens that cause foodborne disease outbreaks and food spoilage, which seriously threatens human health. Bacteriophages have shown broad application prospects in controlling harmful microorganisms during food processing and preservation due to their ability to specifically infect bacteria. In this study, Salmonella enteritidis bacteriophage Salmp-p7 was isolated and characterized from slaughterhouse wastewater. Transmission electron microscopy (TEM) analysis showed that Salmp-p7 belonged to the Siphoviridae family and was active against Salmonella enteritidis and Escherichia coli . Whole genome sequence analysis showed that Salmp-p7 was a lytic bacteriophage with a total length of 60,066 bp of sequence. Salmp-p7 has a short incubation period and a long burst duration, with a burst volume of 55 PFU/cell and a good lysis effect. It can maintain a stable state within the temperature range of 30–60℃ and pH range of 4–12 and has the potential for application in food. In vitro, antimicrobial curves and inhibition of biofilm removal experiments showed that Salmp-p7 could effectively inhibit and eliminate Salmonella enteritidis. The application of Salmp-p7 to the whole liquid of infected eggs resulted in a significant reduction of viable bacteria. And Salmp-p7 has high stability and lytic activity and has the potential to become a new biological control agent for Salmonella enteritidis in eggs .

Listeriosis, caused by Listeria monocytogenes (Lm), is a severe foodborne illness with a high fatality rate. Listeria phages specifically target and lyse Lm, offer a promising alternative for biocontrol and phage therapy. However, most existing studies focus on the lytic characteristics of Listeria phages using limited sample sizes. In this study, a large number of Listeria phages were isolated from diverse sources, and their lytic profiles and morphology were characterized. A total of 317 Listeria phages were isolated from 90 food-related environmental samples and 196 natural environmental samples collected across seven provinces. The phages were tested for lytic activity against 35 Lm strains representing nine serotypes, and their morphology was characterized using transmission electron microscopy (TEM). Statistical analysis was conducted to evaluate the lytic patterns of phages. The phages were classified into three groups based on their total lysis ratios. Broad Host Range Phages (BHRP) were primarily members of the Myoviridae-like phages and demonstrated the ability to lyse a vast majority of nine serotype host strains. Medium Host Range Phages (MHRP) comprised both Siphoviridae-like and Myoviridae-like phages, and demonstrated lysis of 6-9 serotype strains. Narrow Host Range Phages (NHRP) belonged to the Siphoviridae-like phages and exhibited effective lysis of serotype 4 strains. Furthermore, phages isolated from food-related environmental sources demonstrated greater lytic activity against Listeria serotypes 1/2b, 4a, and 4c compared to those derived from natural environmental sources. The study first isolated a multitude of Listeria phages, elucidated their lytic patterns and ecological distribution, and provided a valuable resource for future research.

Background Salmonella enterica serovar Typhimurium is one of the most common serovars of Salmonella associated with clinical cases. It not only leads to diarrhea and mortality raised in livestock and poultry farming, but also poses a risk to food safety. Results In this study, a lytic bacteriophage named ZK22 was isolated and identified from sewage. It exhibited favorable capability against 20 strains of S. Typhimurium. The genome of ZK22 consisted of a double-stranded DNA with a total length of 47,066 base pairs and a GC content of 45.71%. A total of 78 coding sequences were predicted, with no virulence genes or drug resistance genes predicted. Based on blastn similarity analysis, ZK22 belongs to the genus Skatevirus of the class Caudoviricetes, as a long-tailed Siphovirus. Biological characteristics of ZK22 indicated that the optimal multiplicity of infection (MOI) of bacteriophage ZK22 to S. Typhimurium was 10 − 2 (PFU/cell), with an incubation period of around 10 min and the burst size of 393 PFU/cell. The physicochemical resistance results of ZK22 demonstrated that it maintained stability under temperatures ranging from 4 °C to 70 °C and under pH conditions ranging from 3 to 12. After inoculating S. Typhimurium at 37 °C, co-culture mixed with the optimal multiplicity of infection exhibited the lowest OD600 within 12 h, demonstrating the exceptional antibacterial effect. Inoculation of phage ZK22 in mice infected with S. Typhimurium was performed to assess its therapeutic efficacy in vivo. The results showed that phage ZK22 at a dose of 10 8 PFU/mL increased the survival rates of infected mice, effectively suppressed the dissemination and colonization of the host bacteria in the mice, and alleviated the inflammatory response caused by the infection. Conclusions In summary, bacteriophage ZK22 presents favorable application prospects as a potential biological agent for the control of Salmonella infections in livestock and poultry farming.

Background Members of the Roseobacter lineage are a major group of marine heterotrophic bacteria because of their wide distribution, versatile lifestyles and important biogeochemical roles. Bacteriophages, the most abundant biological entities in the ocean, play important roles in shaping their hosts’ population structures and mediating genetic exchange between hosts. However, our knowledge of roseophages (bacteriophages that infect Roseobacter ) is far behind that of their host counterparts, partly reflecting the need to isolate and analyze the phages associated with this ecologically important bacterial clade. Methods vB_DshS-R4C (R4C), a novel virulent roseophage that infects Dinoroseobacter shibae DFL12 T , was isolated with the double-layer agar method. The phage morphology was visualized with transmission electron microscopy. We characterized R4C in-depth with a genomic analysis and investigated the distribution of the R4C genome in different environments with a metagenomic recruitment analysis. Results The double-stranded DNA genome of R4C consists of 36,291 bp with a high GC content of 66.75%. It has 49 genes with low DNA and protein homologies to those of other known phages. Morphological and phylogenetic analyses suggested that R4C is a novel member of the family Siphoviridae and is most closely related to phages in the genus Cronusvirus . However, unlike the Cronusvirus phages, R4C encodes an integrase, implying its ability to establish a lysogenic life cycle. A terminal analysis shows that, like that of λ phage, the R4C genome utilize the ‘cohesive ends’ DNA-packaging mechanism. Significantly, homologues of the R4C genes are more prevalent in coastal areas than in the open ocean. Conclusions Information about this newly discovered phage extends our understanding of bacteriophage diversity, evolution, and their roles in different environments.

Abstract Stenotrophomonas infections pose significant therapeutic challenges due to escalating resistance to antibiotics and chemotherapeutic agents. Phages offer a potential solution by virtue of their specific bacterial targeting capabilities. In this study, we isolated a new Stenotrophomonas bacteriophage, named BUCT627, from hospital sewage. Phage BUCT627 exhibited a 30-min latent period and demonstrated a burst size of 46 plaque forming unit (PFU)/cell. Remarkably, this phage displayed robust stability across a wide pH range (pH 3–13) and exhibited resilience under varying thermal conditions. The receptor of phage BUCT627 on Stenotrophomonas maltophilia No. 826 predominantly consist of surface proteins. The complete genome of phage BUCT627 is a 61 860-bp linear double-stranded DNA molecule with a GC content of 56.3%, and contained 99 open reading frames and two tRNAs. Notably, no antibiotic resistance, toxin, virulence-related genes, or lysogen-formation gene clusters was identified in BUCT627. Transmission electron microscopy and phylogeny analysis indicated that this phage was a new member within the Siphoviridae family. The results of this study will enhance our understanding of phage diversity and hold promise for the development of alternative therapeutic strategies against S. maltophilia infections. A lytic bacteriophage BUCT627 against Stenotrophomonas maltophilia.

This study aimed to isolate and characterize biological and genomic features of a phage infecting Enterococcus faecalis. The phage was isolated from environmental water and temperature and pH stability, one-step growth curve, and multiplicity of infection (MOI) were determined. Whole genome sequencing (WGS) and structural and functional annotations were performed. Its antibiofilm activity was also evaluated. The optimal MOI was 0.01, the latency period was 5 min, and the burst size was 202 plaque forming unit (PFU). High phage survival rates were observed at between pH 4–10 and temperatures between 4–50 °C. WGS and Transmission electron microscopy (TEM) showed that it was an Efquatrovirus representing siphovirus morphotype respectively. It was named as Enterococcus phage Ef212 and has a linear 40,690 bp double-stranded DNA with 45.3% G + C content (GenBank accession number: OR052631). BACPHLIP tool demonstrated that Enterococcus phage Ef212 is a lytic phage (88%). A total of 80 open reading frames (ORFs) were found and there were no antibiotic resistance genes, pathogenicity, virulence genes, or tRNAs in the phage genome. It was diverged from the most similar phages (identity, 88.35%; coverage, 89%) by phylogenetic analysis. Phage Ef212 shared a large part of its genome (60/80) with several other phages, yet some unique parts were found in their genomes. Host range analysis showed that phage Ef212 showed lytic activity against vancomycin-resistant and vancomycin-susceptible E. faecalis clinical isolates. This novel phage Ef212 showed the ability to inhibit and reduce the biofilm formation by around 42% and 38%, respectively. The biological and genomic features indicate that having an effective antibacterial activity, phage Ef212 seemed a promising therapeutic and biocontrol agent.

Bacteriophages are abundant in human biomes and therefore in human clinical samples. Although this is usually not considered, they might interfere with the recovery of bacterial pathogens at two levels: 1) by propagating in the enrichment cultures used to isolate the infectious agent, causing the lysis of the bacterial host and 2) by the detection of bacterial genes inside the phage capsids that mislead the presence of the bacterial pathogen. To unravel these interferences, human samples (n = 271) were analyzed and infectious phages were observed in 11% of blood culture, 28% of serum, 45% of ascitic fluid, 14% of cerebrospinal fluid and 23% of urine samples. The genetic content of phage particles from a pool of urine and ascitic fluid samples corresponded to bacteriophages infecting different bacterial genera. In addition, many bacterial genes packaged in the phage capsids, including antibiotic resistance genes and 16S rRNA genes, were detected in the viromes. Phage interference can be minimized applying a simple procedure that reduced the content of phages up to 3 logs while maintaining the bacterial load. This method reduced the detection of phage genes avoiding the interference with molecular detection of bacteria and reduced the phage propagation in the cultures, enhancing the recovery of bacteria up to 6 logs.

Background Xanthomonas vasicola pv. musacearum is responsible for the widespread Banana Xanthomonas Wilt in banana cultivation regions across the globe. Biocontrol measures for disease management remain limited amidst increasing antimicrobial resistance and unsustainable conventional agricultural practices. The purpose of this study is to explore a viable alternative or adjunct strategy through the use of bacteriophages for disease management. Results Kintu was isolated from sewage and displayed clear and circular plaques measuring 3 mm. Based on transmission electron microscopy, Kintu displays siphovirus characteristics, including an icosahedral head and a non-contractile tail. Kintu infects 78% (22 out of 28) Ugandan Xvm strains, has an optimal multiplicity of infection of 1, a 10 min adsorption and latent period, a 35 min burst period, and a burst size of 15 particles per bacterium. Phage titers remain stable for two and half months (75 days) in SM buffer at -20 o C and − 40 o C but decrease significantly ( p  ≤ 0.0001) at 4 o C. Kintu is active at pH 3 and 11, maintains viability at temperatures between 25 o C and 120 o C and tolerates UV irradiation for up to 2 min and 20 s. Kintu inhibits Xvm growth at MOI ratios of 0.1, 1 and 10. The genome is a double stranded DNA molecule that consists of 48,985 base pairs and a G + C content of 51.71%. Antibiotic resistance genes or genes associated with a lysogenic life cycle are absent. There is limited sequence similarity of Kintu with other phages, making it a novel phage belonging to an unclassified genus of the class Caudoviricetes . Conclusion Kintu is a novel bacteriophage that infects and lyses Xanthomonas vasicola pv. musacearum , the causative agent for Banana Xanthomonas Wilt. Its stability across diverse temperatures and pH conditions highlights its potential as a biocontrol agent for managing the disease.