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The bacterial genus Pseudomonas is a common causative agent of infections in veterinary medicine. In this study, we focused on Pseudomonas aeruginosa canine otitis externa isolates. Due to prolonged antibiotic treatment of otitis externa, antibiotic resistance is common and has become a major complication. Many alternatives to antibiotics have been studied, with bacteriophages emerging as the most promising alternatives. Here, we isolated and characterized a novel phage, pPa_SNUABM_DT01, by investigating its morphology, growth, lysis kinetics, and genomic characteristics. Phages have a vigorous capacity to eliminate bacterial cells through bacterial lysis. This capacity is dependent on the multiplicity of infection (MOI), but even at low MOIs, the phage successfully inhibited bacterial regrowth. The phage genome was 265,520 bp in size and comprised 312 putative open reading frames (ORFs). Comparative genome analysis demonstrated that the phage is a novel species in Myoviridae. The nucleotide similarity was moderately high compared with the Pseudomonas virus, Noxifer. However, a phylogenetic analysis and a dot plot indicated that pPa_SNUABM_DT01 is not closely related to the Phikzvirus or Noxifervirus genus but, instead, belongs to a novel one. The genome comparisons also indicate that the phage, pPa_SNUABM_DT01, could be a novel genus.

Marine viruses are widely distributed and influence matter and energy transformation in ecosystems by modulating hosts’ metabolism. The hadal trenches represent the deepest marine habitat on Earth, for which the viral communities and related biogeochemical functions are least explored and poorly understood. Here, using the sediment samples (8720 m below sea level) collected from the New Britain Trench (NBT), we investigated the viral community, diversity, and genetic potentials in the hadal sediment habitat for the first time by deep shotgun metagenomic sequencing. We found the NBT sediment viral community was dominated by Siphoviridae, Myoviridae, Podoviridae, Mimiviridae, and Phycodnaviridae, which belong to the dsDNA viruses. However, the large majority of them remained uncharacterized. We found the hadal sediment virome had some common components by comparing the hadal sediment viruses with those of hadal aquatic habitats and those of bathypelagic and terrestrial habitats. It was also distinctive in community structure and had many novel viral clusters not associated with the other habitual virome included in our analyses. Further phylogenetic analysis on its Caudovirales showed novel diversities, including new clades specially evolved in the hadal sediment habitat. Annotation of the NBT sediment viruses indicated the viruses might influence microbial hydrocarbon biodegradation and carbon and sulfur cycling via metabolic augmentation through auxiliary metabolic genes (AMGs). Our study filled in the knowledge gaps on the virome of the hadal sediment habitats and provided insight into the evolution and the potential metabolic functions of the hadal sediment virome.

Due to the increasing spread of multidrug-resistant (MDR) bacteria, phage therapy is considered one of the most promising methods for addressing MDR bacteria. Escherichia coli lives symbiotically in the intestines of humans and some animals, and most strains are beneficial in terms of maintaining a healthy digestive tract. However, some E. coli strains can cause serious zoonotic diseases, including diarrhea, pneumonia, urinary tract infections, and hemolytic uremic syndrome. In this study, we characterized a newly isolated Myoviridae phage, vB_EcoM_APEC. The phage vB_EcoM_APEC was able to infect E. coli APEC O78, which is the most common MDR E. coli serotype in turkeys. Additionally, the phage’s host range included Klebsiella pneumoniae and other E. coli strains. The genome of phage vB_EcoM_APEC (GenBank accession number MT664721) was 35,832 bp in length, with 52 putative open reading frames (ORFs) and a GC content of 41.3%. The genome of vB_EcoM_APEC exhibited low similarity (79.1% identity and 4.0% coverage) to the genome of Acinetobacter phage vB_AbaM_IME284 (GenBank no. MH853787.1) according to the nucleotide Basic Local Alignment Search Tool (BLASTn). Phylogenetic analysis revealed that vB_EcoM_APEC was a novel phage, and its genome sequence showed low similarity to other available phage genomes. Gene annotation indicated that the protein encoded by orf11 was an endolysin designated as LysO78, which exhibited 64.7% identity (91.0% coverage) with the putative endolysin of Acinetobacter baumannii phage vB_AbaM_B9. The LysO78 protein belongs to glycoside hydrolase family 19, and was described as being a chitinase class I protein. LysO78 is a helical protein with 12 α-helices containing a large domain and a small domain in terms of the predicted three-dimensional structure. The results of site-directed mutagenesis indicated that LysO78 contained the catalytic residues E54 and E64. The purified endolysin exhibited broad-spectrum bacteriolytic activity against Gram-negative strains, including the genera Klebsiella, Salmonella, Shigella, Burkholderia, Yersinia, and Pseudomonas, as well as the species Chitinimonas arctica, E. coli, Ralstonia solanacearum, and A. baumannii. An enzymatic assay showed that LysO78 had highly lytic peptidoglycan hydrolases activity (64,620,000 units/mg) against E. coli APEC O78, and that LysO78 had lytic activity in the temperature range of 4–85 °C, with an optimal temperature of 28 °C and optimal pH of 8.0, and was active at pH 3.0–12.0. Overall, the results suggested that LysO78 might be a promising therapeutic agent for controlling MDR E. coli APEC O78 and nosocomial infections caused by multidrug-resistant bacteria.

Sulfate-reducing bacteria (SRB) represent a group of prokaryotic microorganisms that are widely spread in the anoxic environment (seabed, riverbed and lakebed sediments, mud, intestinal tract of humans and animals, metal surfaces). SRB species also have an impact on processes occurring in the intestinal tract of humans and animals, including the connections between their presence and inflammatory bowel disease (IBD). Since these SRB can develop antimicrobial resistance toward the drugs, including antibiotics and antimicrobial agents, bacteriophages could represent an additional potential effective treatment. The main objectives of the review were as follows: (a) to review SRB (both from intestinal and environmental sources) regarding their role in intestinal diseases as well as their influence in environmental processes; and (b) to review, according to literature data, the influence of bacteriophages on SRB and their possible applications. Since SRB can have a significant adverse influence on industry as well as on humans and animals health, phage treatment of SRB can be seen as a possible effective method of SRB inhibition. However, there are relatively few studies concerning the influence of phages on SRB strains. Siphoviridae and Myoviridae families represent the main sulfide-producing bacteria phages. The most recent studies induced, by UV light, bacteriophages from Desulfovibrio vulgaris NCIMB 8303 and Desulfovibrio desulfuricans ATCC 13541. Notwithstanding costly and medically significant negative impacts of phages on SRB, they have been the subject of relatively few studies. The current search for alternatives to chemical biocides and antibiotics has led to the renewed interest in phages as antibacterial biocontrol and therapeutic agents, including their use against SRB. Hence, phages might represent a promising treatment against SRB in the future.

The abundant and widespread unicellular cyanobacteria Synechococcus plays an important role in contributing to global phytoplankton primary production. In the present study, two novel cyanomyoviruses, S-N03 and S-H34 that infected Synechococcus MW02, were isolated from the coastal waters of the Yellow Sea. S-N03 contained a 167,069-bp genome comprising double-stranded DNA with a G + C content of 50.1%, 247 potential open reading frames and 1 tRNA, S-H34 contained a 167,040-bp genome with a G + C content of 50.1%, 246 potential open reading frames and 5 tRNAs. These two cyanophages contain fewer auxiliary metabolic genes (AMGs) than other previously isolated cyanophages. S-H34 in particular, is currently the only known cyanomyovirus that does not contain any AMGs related to photosynthesis. The absence of such common AMGs in S-N03 and S-H34，their distinct evolutionary history and ecological features imply that the energy for phage production might be obtained from other sources rather than being strictly dependent on the maintenance of photochemical ATP under high light. Phylogenetic analysis showed that the two isolated cyanophages clustered together and had a close relationship with two other cyanophages of low AMG content. Comparative genomic analysis, habitats and hosts across 81 representative cyanomyovirus showed that cyanomyovirus with less AMGs content all belonged to Synechococcus phages isolated from eutrophic waters. The relatively small genome size and high G + C content may also relate to the lower AMG content, as suggested by the significant correlation between the number of AMGs and G + C%. Therefore, the lower content of AMG in S-N03 and S-H34 might be a result of viral evolution that was likely shaped by habitat, host, and their genomic context. The genomic content of AMGs in cyanophages may have adaptive significance and provide clues to their evolution.

Bacteriophage PaBG is a jumbo Myoviridae phage isolated from water of Lake Baikal. This phage has limited diffusion ability and thermal stability and infects a narrow range of Pseudomonas aeruginosa strains. Therefore, it is hardly suitable for phage therapy applications. However, the analysis of the genome of PaBG presents a number of insights into the evolutionary history of this phage and jumbo phages in general. We suggest that PaBG represents an ancient group distantly related to all known classified families of phages.

Mortalities of bivalve larvae and spat linked with Vibrio spp. infection have been described in hatcheries since 1959, causing potential development of resistant bacteria. A reliable and sustainable solution to this problem is yet to be developed. Potential treatment of bacterial infection with bacteriophages is gaining interest in aquaculture as a more sustainable option for managing Vibrio spp. infection. This study assessed the effectiveness of bacteriophages (&Phi, 5, &Phi, 6, and &Phi, 7) against pathogenic Vibrio isolates (USC-26004 and USC-26005). These phage isolates were found to belong to the Myoviridae viral family. A total of 212 ORFs of &Phi, 5 were identified and annotated. The genome of this phage contained putative thymidine kinase and lysin enzyme. During infections with phages, the OD values of the isolates USC-26005 and USC-26004 remained stable at a much lower reading compared to the control after 9 h of incubation. Mortality rate of oyster (Saccostrea glomerata) larvae was 28.2 &plusmn, 3.5% in the bacteriophage treatment group, compared to 77.9 &plusmn, 9.1% in the bacterial treatment group after 24 h incubation. Findings of this study indicate that lytic phages might be utilized as potential bio-control agents of luminescent bacterial disease in oyster hatcheries.

Bacterial fruit blotch caused by Acidovorax citrulli is known to be the major threat to cucurbit crop production worldwide. The pathogen can penetrate into seed coat and cause disease symptoms at any stage of plant growth, which results in fruit loss. Two main genotypes (genotype I and II) are reported in A. citrulli, in which genotype II is the main cause of Bacterial Fruit Blotch (BFB) in watermelon and group I is known to be a causal agent of BFB in melon. To date, there are no commercially available cultivars resistant to BFB, and available strategies are not able to completely manage the disease. In this study, we aim to isolate bacteriophages to control BFB. Samples collected from watermelon, melon, and pumpkin were used to isolate bacteriophages. All isolated bacteriophages were tested against 42 strains of A. citrulli, among which two phages with the ability to lyse a greater number of hosts were selected and characterized. Bacteriophage ACP17 from the Myoviridae family, with a head size of 100 ± 5 nm and tail of 150 ± 5 nm, infected 29 strains of A. citrulli mostly belonging to genotype group I, whereas the second isolated bacteriophage, ACPWH from Siphoviridae, with a head size of 60 ± 5 nm and tail of 180 ± 5 nm, infected 39 A. citrulli strains. Genome analysis of both bacteriophages using Next generation Sequencing (NGS) showed that ACP17 and ACPWH have double-stranded DNA with sizes of 156,972 kb and 424,299 kb, respectively. Watermelon seeds coated with ACPWH showed a germination rate of up to 90% in the presence of A. citrulli in contrast to untreated seed, which showed no germination or germinated juveniles with BFB symptoms in the presence of A. citrulli. The results of this study show that the use of bacteriophages of A. citrulli represents a potential biocontrol method for controlling BFB.

Salmonella is a leading cause of foodborne diseases, and in recent years, many isolates have exhibited a high level of antibiotic resistance, which has led to huge pressures on public health. Phages are a promising strategy to control food-borne pathogens. In this study, one of our environmental phage isolates, LPSEYT, was to be able to restrict the growth of zoonotic Salmonella enterica in vitro over a range of multiplicity of infections. Phage LPSEYT exhibited wide-ranging pH and thermal stability and rapid reproductive activity with a short latent period and a large burst size. Phage LPSEYT demonstrated potential efficiency as a biological control agent against Salmonella in a variety of food matrices, including milk and lettuce. Morphological observation, comparative genomic, and phylogenetic analysis revealed that LPSEYT does not belong to any of the currently identified genera within the Myoviridae family, and we suggest that LPSEYT represents a new genus, the LPSEYTvirus. This study contributes a phage database, develops beneficial phage resources, and sheds light on the potential application value of phages LPSEYT on food safety.

Klebsiella pneumoniae is an increasing threat to public health and represents one of the most concerning pathogens involved in life-threatening infections. The resistant and virulence determinants are coded by mobile genetic elements which can easily spread between bacteria populations and co-evolve with its genomic host. In this study, we present the full genomic sequences, insertion sites and phylogenetic analysis of 150 prophages found in 40 K. pneumoniae clinical isolates obtained from an outbreak in a Portuguese hospital. All strains harbored at least one prophage and we identified 104 intact prophages (69.3%). The prophage size ranges from 29.7 to 50.6 kbp, coding between 32 and 78 putative genes. The prophage GC content is 51.2%, lower than the average GC content of 57.1% in K. pneumoniae. Complete prophages were classified into three families in the order Caudolovirales: Myoviridae (59.6%), Siphoviridae (38.5%) and Podoviridae (1.9%). In addition, an alignment and phylogenetic analysis revealed nine distinct clusters. Evidence of recombination was detected within the genome of some prophages but, in most cases, proteins involved in viral structure, transcription, replication and regulation (lysogenic/lysis) were maintained. These results support the knowledge that prophages are diverse and widely disseminated in K. pneumoniae genomes, contributing to the evolution of this species and conferring additional phenotypes. Moreover, we identified K. pneumoniae prophages in a set of endolysin genes, which were found to code for proteins with lysozyme activity, cleaving the β-1,4 linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in the peptidoglycan network and thus representing genes with the potential for lysin phage therapy., F.F.V. is funded by Fundação para a Ciência e a Tecnologia (FCT) through an Assistant Researcher grant CEECIND/03023/2017, and a project grant (PTDC/BTM-SAL/28978/2017) that supported this work. The work is partially supported by National funds from FCT, projects UIDB/04138/2020 and UIDP/04138/2020.

Phage G is recognized as having a remarkably large genome and capsid size among isolated, propagated phages. Negative stain electron microscopy of the host–phage G interaction reveals tail sheaths that are contracted towards the distal tip and decoupled from the head–neck region. This is different from the typical myophage tail contraction, where the sheath contracts upward, while being linked to the head–neck region. Our cryo-EM structures of the non-contracted and contracted tail sheath show that: (1) The protein fold of the sheath protein is very similar to its counterpart in smaller, contractile phages such as T4 and phi812, (2) Phage G’s sheath structure in the non-contracted and contracted states are similar to phage T4’s sheath structure. Similarity to other myophages is confirmed by a comparison-based study of the tail sheath’s helical symmetry, the sheath protein’s evolutionary timetree, and the organization of genes involved in tail morphogenesis . Atypical phase G tail contraction could be due to a missing anchor point at the upper end of the tail sheath that allows the decoupling of the sheath from the head­–neck region. Explaining the atypical tail contraction requires further investigation of the phage G sheath anchor points.

Enterotoxigenic Escherichia coli (ETEC) and Shigella ssp. infections are associated with high rates of mortality, especially in infants in developing countries. Due to increasing levels of global antibiotic resistance exhibited by many pathogenic organisms, alternative strategies to combat such infections are urgently required. In this study, we evaluated the stability of five coliphages (four Myoviridae and one Siphoviridae phage) over a range of pH conditions and in simulated gastric conditions. The Myoviridae phages were stable across the range of pH 2 to 7, while the Siphoviridae phage, JK16, exhibited higher sensitivity to low pH. A composite mixture of these five phages was tested in vivo in a Galleria mellonella model. The obtained data clearly shows potential in treating E. coli infections prophylactically.

Bacteriophages of thermophiles are of increasing interest owing to their important roles in many biogeochemical, ecological processes and in biotechnology applications, including emerging bionanotechnology. However, due to lack of in-depth investigation, they are underrepresented in the known prokaryotic virosphere. Therefore, there is a considerable potential for the discovery of novel bacteriophage-host systems in various environments: marine and terrestrial hot springs, compost piles, soil, industrial hot waters, among others. This review aims at providing a reference compendium of thermophages characterized thus far, which infect the species of thermophilic ‘Bacillus group’ bacteria, mostly from Geobacillus sp. We have listed 56 thermophages, out of which the majority belong to the Siphoviridae family, others belong to the Myoviridae and Podoviridae families and, apparently, a few belong to the Sphaerolipoviridae, Tectiviridae or Corticoviridae families. All of their genomes are composed of dsDNA, either linear, circular or circularly permuted. Fourteen genomes have been sequenced; their sizes vary greatly from 35,055 bp to an exceptionally large genome of 160,590 bp. We have also included our unpublished data on TP-84, which infects Geobacillus stearothermophilus (G. stearothermophilus). Since the TP-84 genome sequence shows essentially no similarity to any previously characterized bacteriophage, we have defined TP-84 as a new species in the newly proposed genus Tp84virus within the Siphoviridae family. The information summary presented here may be helpful in comparative deciphering of the molecular basis of the thermophages’ biology, biotechnology and in analyzing the environmental aspects of the thermophages’ effect on the thermophile community.

This study describes two viral communities from the world&rsquo, s oldest lake, Lake Baikal. For the analysis, we chose under-ice and late spring periods of the year as the most productive for Lake Baikal. These periods show the maximum seasonal biomass of phytoplankton and bacterioplankton, which are targets for viruses, including bacteriophages. At that time, the main group of viruses were tailed bacteriophages of the order Caudovirales that belong to the families Myoviridae, Siphoviridae and Podoviridae. Annotation of functional genes revealed that during the under-ice period, the &ldquo, Phages, Prophages, Transposable Elements and Plasmids&rdquo, (27.4%) category represented the bulk of the virome. In the late spring period, it comprised 9.6% of the virome. We assembled contigs by two methods: Separately assembled in each virome or cross-assembled. A comparative analysis of the Baikal viromes with other aquatic environments indicated a distribution pattern by soil, marine and freshwater groups. Viromes of lakes Baikal, Michigan, Erie and Ontario form the joint World&rsquo, s Largest Lakes clade.

Jumbo phages, which have a genome size of more than 200 kb, have recently been reported for the first time. However, limited information is available regarding their characteristics because few jumbo phages have been isolated. Therefore, in this study, we aimed to isolate and characterize other jumbo phages. We performed comparative genomic analysis of three Erwinia phages (pEa_SNUABM_12, pEa_SNUABM_47, and pEa_SNUABM_50), each of which had a genome size of approximately 360 kb (32.5% GC content). These phages were predicted to harbor 546, 540, and 540 open reading frames with 32, 34, and 35 tRNAs, respectively. Almost all of the genes in these phages could not be functionally annotated but showed high sequence similarity with genes encoded in Serratia phage BF, a member of Eneladusvirus. The detailed comparative and phylogenetic analyses presented in this study contribute to our understanding of the diversity and evolution of Erwinia phage and the genus Eneladusvirus.

Bacteriophages and phage enzymes are considered as possible alternatives to antibiotics in the treatment of infections caused by antibiotic-resistant bacteria. Due to the ability to cleave the capsular polysaccharides (CPS), one of the main virulence factors of Klebsiella pneumoniae, phage depolymerases, has potential in the treatment of K. pneumoniae infections. Here, we characterized in vivo two novel phage-encoded polysaccharide depolymerases as therapeutics against clinical isolates of K. pneumoniae. The depolymerases Dep_kpv79 and Dep_kpv767 encoded by Klebsiella phages KpV79 (Myoviridae, Jedunavirus) and KpV767 (Autographiviridae, Studiervirinae, Przondovirus), respectively, were identified as specific &beta, galactosidases that cleave the K. pneumoniae K57 type CPS by the hydrolytic mechanism. They were found to be highly effective at combating sepsis and hip infection caused by K. pneumoniae in lethal mouse models. Here, 80&ndash, 100% of animals were protected against death by a single dose (e.g., 50 &mu, g/mouse) of the enzyme injected 0.5 h after infection by K. pneumoniae strains of the K57 capsular type. The therapeutic effect of the depolymerases is because they strip the capsule and expose the underlying bacterium to the immune attack such as complement-mediated killing. These data provide one more confirmation that phage polysaccharide depolymerases represent a promising tool for antimicrobial therapy.

Phage stability is important for the successful application of bacteriophages as alternative antibacterial agents. Considering that temperature is a critical factor in phage stability, this study aimed to explore the possibility of improving long-term phage stability through adaptive evolution to elevated temperature. Evolution of three wild-type ancestral phages (Myoviridae phage Wc4 and Podoviridae phages CX5 and P-PSG-11) was induced by subjecting the phages to heat treatment at 60 &deg, C for five cycles. The adapted phages showed better stability than the wild-type ancestral phages when subjected to heat treatment at 60 &deg, C for 1 h and after 60 days of storage at 37 &deg, C. However, the adapted phages could not withstand thermal treatment at 70 &deg, C for 1 h. The infectivity and the lytic properties of the phages were not changed by the evolution process. Whole-genome sequencing revealed that single substitutions in the tail tubular proteins were the only changes observed in the genomes of the adapted phages. This study demonstrates that adaptive evolution could be used as a general method for enhancing the thermal stability of phages without affecting their lytic activity. Sequencing results showed that bacteriophages may exist as a population with minor heterogeneous mutants, which might be important to understand the ecology of phages in different environments.

Resistance to carbapenems in Enterobacteriaceae, including Klebsiella pneumoniae, represents a major clinical problem given the lack of effective alternative antibiotics. Bacteriophages could provide a valuable tool to control the dissemination of antibiotic resistant isolates, for the decolonization of colonized individuals and for treatment purposes. In this work, we have characterized a lytic bacteriophage, named vB_Kpn_F48, specific for K. pneumoniae isolates belonging to clonal group 101. Phage vB_Kpn_F48 was classified as a member of Myoviridae, order Caudovirales, on the basis of transmission electron microscopy analysis. Physiological characterization demonstrated that vB_Kpn_F48 showed a narrow host range, a short latent period, a low burst size and it is highly stable to both temperature and pH variations. High throughput sequencing and bioinformatics analysis revealed that the phage is characterized by a 171 Kb dsDNA genome that lacks genes undesirable for a therapeutic perspective such integrases, antibiotic resistance genes and toxin encoding genes. Phylogenetic analysis suggests that vB_Kpn_F48 is a T4-like bacteriophage which belongs to a novel genus within the Tevenvirinae subfamily, which we tentatively named &ldquo, F48virus&rdquo, Considering the narrow host range, the genomic features and overall physiological parameters phage vB_Kpn_F48 could be a promising candidate to be used alone or in cocktails for phage therapy applications.

To determine the effectivity of bacteriophages in controlling the mass mortality of striped catfish (Pangasianodon hypophthalmus) due to infections caused by Aeromonas spp. in Vietnamese fish farms, bacteriophages against pathogenic Aeromonas hydrophila were isolated. A. hydrophila-phage 2 and A. hydrophila-phage 5 were successfully isolated from water samples from the Saigon River of Ho Chi Minh City, Vietnam. These phages, belonging to the Myoviridae family, were found to have broad activity spectra, even against the tested multiple-antibiotic-resistant Aeromonas isolates. The latent periods and burst size of phage 2 were 10 min and 213 PFU per infected host cell, respectively. The bacteriophages proved to be effective in inhibiting the growth of the Aeromonas spp. under laboratory conditions. Phage treatments applied to the pathogenic strains during infestation of catfish resulted in a significant improvement in the survival rates of the tested fishes, with up to 100% survival with MOI 100, compared to 18.3% survival observed in control experiments. These findings illustrate the potential for using phages as an effective bio-treatment method to control Motile Aeromonas Septicemia (MAS) in fish farms. This study provides further evidence towards the use of bacteriophages to effectively control disease in aquaculture operations.

Pseudomonas aeruginosa is one of the Multi-Drug-Resistant organisms most frequently isolated worldwide and, because of a shortage of new antibiotics, bacteriophages are considered an alternative for its treatment. Previously, P. aeruginosa phages were isolated and best candidates were chosen based on their ability to form clear plaques and their host range. This work aimed to characterize one of those phages, ΦPan70, preliminarily identified as a good candidate for phage-therapy. We performed infection curves, biofilm removal assays, transmission-electron-microscopy, pulsed-field-gel-electrophoresis, and studied the in vivo ΦPan70 biological activity in the burned mouse model. ΦPan70 was classified as a member of the Myoviridae family and, in both planktonic cells and biofilms, was responsible for a significant reduction in the bacterial population. The burned mouse model showed an animal survival between 80% and 100%, significantly different from the control animals (0%). However, analysis of the ΦPan70 genome revealed that it was 64% identical to F10, a temperate P. aeruginosa phage. Gene annotation indicated ΦPan70 as a new, but possible temperate phage, therefore not ideal for phage-therapy. Based on this, we recommend genome sequence analysis as an early step to select candidate phages for potential application in phage-therapy, before entering into a more intensive characterization.

The Spounavirinae viruses are ubiquitous in nature and have an obligatory virulent lifestyle. They infect Firmicutes, a bacterial phylum containing an array of environmental non-pathogenic and pathogenic organisms. To expand the knowledge of this viral subfamily, new strains were isolated and investigated in this study. Here we present two new viruses, vB_BsuM-Goe2 and vB_BsuM-Goe3, isolated from raw sewage and infecting Bacillus species. Both were morphologically classified via transmission electron microscopy (TEM) as members of the Spounavirinae subfamily belonging to the Myoviridae family. Genomic sequencing and analyses allowed further affiliation of vB_BsuM-Goe2 to the SPO1-like virus group and vB_BsuM-Goe3 to the Bastille-like virus group. Experimentally determined adsorption constant, latency period, burst size and host range for both viruses revealed different survival strategies. Thus vB_BsuM-Goe2 seemed to rely on fewer host species compared to vB_BsuM-Goe3, but efficiently recruits those. Stability tests pointed out that both viruses are best preserved in LB-medium or TMK-buffer at 4 or 21 °C, whereas cryopreservation strongly reduced viability.

In the rhizosphere, bacteria&ndash, phage interactions are likely to have important impacts on the ecology of microbial communities and microbe&ndash, plant interactions. To better understand the dynamics of Agrobacteria&ndash, phage interactions, we have isolated diverse bacteriophages which infect the bacterial plant pathogen, Agrobacterium tumefaciens. Here, we complete the genomic characterization of Agrobacterium tumefaciens phages Atu_ph04 and Atu_ph08. Atu_ph04&mdash, a T4-like phage belonging to the Myoviridae family&mdash, was isolated from waste water and has a 143,349 bp genome that encodes 223 predicted open reading frames (ORFs). Based on phylogenetic analysis and whole-genome alignments, Atu_ph04 is a member of a newly described T4 superfamily that contains other Rhizobiales-infecting phages. Atu_ph08, a member of the Podoviridae T7-like family, was isolated from waste water, has a 59,034 bp genome, and encodes 75 ORFs. Based on phylogenetic analysis and whole-genome alignments, Atu_ph08 may form a new T7 superfamily which includes Sinorhizobium phage PCB5 and Ochrobactrum phage POI1126. Atu_ph08 is predicted to have lysogenic activity, as we found evidence of an integrase and several transcriptional repressors with similarity to proteins in transducing phage P22. Together, this data suggests that Agrobacterium phages are diverse in morphology, genomic content, and lifestyle.

The sophisticated antibiotic resistance mechanism of Pseudomonas aeruginosa has urged the development of alternative antibacterial strategies. Phage therapy has been proven successful for the treatment of multidrug-resistant infections. In this study, we reported two virulent P. aeruginosa phages, vB_PaeM_SCUT-S1 (S1) and vB_PaeM_SCUT-S2 (S2), which were characterized at morphological, genomic, and proteomic levels. Phages S1 and S2 were assigned to the Myoviridae family. The genome sequencing showed that the genome size of Phage S1 was 66,046 bp and that of Phage S2 was 94,434 bp. The phylogenetic tree indicated that the two phages were distantly related to each other and were classified in the genera Pbunavirus and Pakpunavirus respectively. Thirty-one proteins were identified for each phage by mass spectrometry and were used to substantiate the function of the predicted coding genes. The two phages inhibited the growth of P. aeruginosa strain PAO1 at low multiplicity of infection levels and had good performance both on preventing biofilm formation and eradicating preformed biofilms. They were also stable over a wide range of temperature and pH values, supporting their potential use in the treatment of P. aeruginosa infections.

The &lsquo, Appelmans protocol&rsquo, is used by Eastern European researchers to generate therapeutic phages with novel lytic host ranges. Phage cocktails are iteratively grown on a suite of mostly refractory bacterial isolates until the evolved cocktail can lyse the phage-resistant strains. To study this process, we developed a modified protocol using a cocktail of three Pseudomonas phages and a suite of eight phage-resistant (including a common laboratory strain) and two phage-sensitive Pseudomona aeruginosa strains. After 30 rounds of selection, phages were isolated from the evolved cocktail with greatly increased host range. Control experiments with individual phages showed little host-range expansion, and genomic analysis of one of the broad-host-range output phages showed its recombinatorial origin, suggesting that the protocol works predominantly via recombination between phages. The Appelmans protocol may be useful for evolving therapeutic phage cocktails as required from well-defined precursor phages.

The Arctic marine environment experiences dramatic seasonal changes in light and nutrient availability. To investigate the influence of seasonality on Arctic marine virus communities, five research cruises to the west and north of Svalbard were conducted across one calendar year, collecting water from the surface to 1000 m in depth. We employed metabarcoding analysis of major capsid protein g23 and mcp genes in order to investigate T4-like myoviruses and large dsDNA viruses infecting prokaryotic and eukaryotic picophytoplankton, respectively. Microbial abundances were assessed using flow cytometry. Metabarcoding results demonstrated that seasonality was the key mediator shaping virus communities, whereas depth exerted a diversifying effect within seasonal virus assemblages. Viral diversity and virus-to-prokaryote ratios (VPRs) dropped sharply at the commencement of the spring bloom but increased across the season, ultimately achieving the highest levels during the winter season. These findings suggest that viral lysis may be an important process during the polar winter, when productivity is low. Furthermore, winter viral communities consisted of Operational Taxonomic Units (OTUs) distinct from those present during the spring-summer season. Our data provided a first insight into the diversity of viruses in a hitherto undescribed marine habitat characterized by extremes in light and productivity.

Bacteriophage (phage) therapy is a promising alternative to antibiotics for the treatment of bacterial pathogens, including Clostridium difficile. However, as for many species, in C. difficile the physical interactions between phages and bacterial cells have not been studied in detail. The initial interaction, known as phage adsorption, is initiated by the reversible attachment of phage tail fibers to bacterial cell surface receptors followed by an irreversible binding step. Therefore binding can dictate which strains are infected by the phage. In this study, we investigated the adsorption rates and irreversible binding of three C. difficile myoviruses: CDHM1, CDHM3 and CDHM6 to ten strains that represent ten prevalent C. difficile ribotypes, regardless of their ability to infect. CDHM1 and CDHM3 phage particles adsorbed by ~75% to some strains that they infected. The infection dynamics for CDHM6 are less clear and ~30% of the phage particles bound to all strains, irrespective of whether a successful infection was established. The data highlighted adsorption is phage-host specific. However, it was consistently observed that irreversible binding had to be above 80% for successful infection, which was also noted for another two C. difficile myoviruses. Furthermore, to understand if there is a relationship between infection, adsorption and phage tail fibers, the putative tail fiber protein sequences of CDHM1, CDHM3 and CDHM6 were compared. The putative tail fiber protein sequence of CDHM1 shares 45% homology at the amino acid level to CDHM3 and CDHM6, which are identical to each other. However, CDHM3 and CDHM6 display differences in adsorption, which highlights that there is no obvious relationship between putative tail fiber sequence and adsorption. The importance of adsorption and binding to successful infection is often overlooked, and this study provides useful insights into host-pathogen interactions within this phage-pathogen system.