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.

Analysis of a new phage, KZag1, infecting biofilm of Klebsiella pneumoniae: genome sequence and characterization.

BACKGROUND: This study investigates the effectiveness of the bacteriophage KZag1 against drug-resistant Klebsiella pneumoniae, aiming to assess its potential as a therapeutic agent. The novelty lies in the characterization of KZag1, a Myovirus with specific efficacy against multidrug-resistant K. pneumoniae strains. This highlights the significance of exploring alternative strategies, particularly phage therapy, in addressing biofilm-associated infections. METHODS: KZag1, characterized by a typical Myovirus structure with a 75 ± 5 nm diameter icosahedral head and a 15 ± 5 nm short tail, was evaluated in experimental trials against 15 strains of K. pneumoniae. The infection cycle duration was determined to be 50 min, resulting in an estimated burst size of approximately 83 plaque-forming units per colony-forming unit (PFU/CFU). Stability assessments were conducted within a pH range of 4 to 12 and temperatures ranging from 45°C to 60°C. Biofilm biomass reduction was observed, particularly at a multiplicity of infection (MOI) of 10. RESULTS: KZag1 demonstrated infection efficacy against 12 out of 15 tested K. pneumoniae strains. The phage exhibited stability across a broad pH range and at elevated temperatures. Notably, treatment with KZag1 significantly reduced K. pneumoniae biofilm biomass, emphasizing its potential in combating biofilm formation. Genomic analysis revealed a complete genome of 157,089 base pairs with a GC content of 46.38%, encompassing 203 open reading frames (ORFs) and a cysteine-specific tRNA sequence. Comparison with phage GP4 highlighted similarities, with KZag1 having a longer genome by approximately 4829 base pairs and a higher GC content by approximately 0.93%. Phylogenetic analysis classified KZag1 within the Myoviridae family. CONCLUSION: The efficacy of KZag1 against K. pneumoniae biofilm suggests its potential as a therapeutic candidate, especially for drug-resistant infections. Further clinical research is warranted to explore its synergy with other treatments, elucidate genomic traits, compare with Myoviridae phages, and understand its host interactions. These findings underscore the promising role of KZag1 in addressing drug-resistant bacterial infections.

In vitro evaluation of two novel Escherichia bacteriophages against multiple drug resistant avian pathogenic Escherichia coli.

BACKGROUND: In recent years, there has been a growing interest in phage therapy as an effective therapeutic tool against colibacillosis caused by avian pathogenic Escherichia coli (APEC) which resulted from the increasing number of multidrug resistant (MDR) APEC strains. METHODS: In the present study, we reported the characterization of a new lytic bacteriophage (Escherichia phage AG- MK-2022. Basu) isolated from poultry slaughterhouse wastewater. In addition, the in vitro bacteriolytic activity of the newly isolated phage (Escherichia phage AG- MK-2022. Basu) and the Escherichia phage VaT-2019a isolate PE17 (GenBank: MK353636.1) were assessed against MDR- APEC strains (n = 100) isolated from broiler chickens with clinical signs of colibacillosis. RESULTS: Escherichia phage AG- MK-2022. Basu belongs to the Myoviridae family and exhibits a broad host range. Furthermore, the phage showed stability under a wide range of temperatures, pH values and different concentrations of NaCl. Genome analysis of the Escherichia phage AG- MK-2022. Basu revealed that the phage possesses no antibiotic resistance genes (ARGs), mobile genetic elements (MGEs), and any E. coli virulence associated genes. In vitro bacterial challenge tests demonstrated that two phages, the Escherichia phage VaT-2019a isolate PE17 and the Escherichia phage AG- MK-2022. Basu exhibited high bactericidal activity against APEC strains and lysed 95% of the tested APEC strains. CONCLUSIONS: The current study findings indicate that both phages could be suggested as safe biocontrol agents and alternatives to antibiotics for controlling MDR-APEC strains isolated from broilers.

T4-like myovirus community shaped by dispersal and deterministic processes in the South China Sea.

As the most abundant and genetically diverse biological entities, viruses significantly influence ecological, biogeographical and evolutionary processes in the ocean. However, the biogeography of marine viruses and the drivers shaping viral community are unclear. Here, the biogeographic patterns of T4-like viruses and the relative impacts of deterministic (environmental selection) and dispersal (spatial distance) processes were investigated in the northern South China Sea. The dominant viral operational taxonomic units were affiliated with previously defined Marine, Estuary, Lake and Paddy Groups. A clear viral biogeographic pattern was observed along the environmental gradient from the estuary to open sea. Marine Groups I and IV had a wide geographical distribution, whereas Marine Groups II, III and V were abundant in lower-salinity continental or eutrophic environments. A significant distance-decay pattern was noted for the T4-like viral community, especially for those infecting cyanobacteria. Both deterministic and dispersal processes influenced viral community assembly, although environmental selection (e.g. temperature, salinity, bacterial abundance and community, etc.) had a greater impact than spatial distance. Network analysis confirmed the strong association between viral and bacterial community composition, and suggested a diverse ecological relationship (e.g. lysis, co-infection or mutualistic) between and within viruses and their potential bacterial hosts.

Genome analysis of Salmonella phage vB_SalM_8-19 (genus Rosemountvirus).

This work describes the characterization and genome annotation of Salmonella phage vB_SalM_8-19 (referred to as 8-19) isolated from sewage samples collected in a pig farm in Jilin, China. This phage was capable of infecting 60% Salmonella strains in our lab stock. The genome of phage 8-19 is composed of linear double-stranded DNA that is 52,648 bp in length with a G + C content of 46.02%; containing 74 ORFs and no tRNA genes. In October 2019, phylogenetic analyses indicated that phage 8-19 might belong to a novel cluster among the other similar phages which have not been specifically classified within some new genus in family Myoviridae. Recently, the International Committee on Taxonomy of Viruses (ICTV) defined phage 8-19 and its related phages as genus Rosemountvirus, family Myoviridae. This new genus, known as Rosemountvirus, is rarely reported in the literature.

Complete genome analysis of the newly isolated Shigella sonnei phage vB_SsoM_Z31.

This work describes the characterization and genome annotation of the newly isolated lytic phage vB_SsoM_Z31 (referred to as Z31), isolated from wastewater samples collected in Dalian, China. Transmission electron microscopy revealed that phage Z31 belongs to the family Myoviridae, order Caudovirales. This phage specifically infects Shigella sonnei, Shigella dysenteriae, and Escherichia coli. The genome of the phage Z31 is an 89,355-bp-long dsDNA molecule with a G+C content of 38.87%. It was predicted to contain 133 ORFs and encode 24 tRNAs. No homologs of virulence factor genes or antimicrobial resistance genes were found in this phage. Based on the results of nucleotide sequence alignment and phylogenetic analysis, phage Z31 was assigned to the genus Felixounavirus, subfamily Ounavirinae.

Characterization and genome sequence of the genetically unique Escherichia bacteriophage vB_EcoM_IME392.

In this study, a novel Escherichia coli-specific bacteriophage, vB_EcoM_IME392, was isolated from chicken farm sewage in Qingdao, China. The genome of IME392 was found by next-generation sequencing to be 116,460 base pairs in length with a G+C content of 45.4% (GenBank accession number MH719082). BLASTn results revealed that only 2% of the genome sequence of IME392 shows sequence similarity to known phage sequences in the GenBank database, which indicates that IME392 is a novel bacteriophage. Transmission electron microscopy showed that IME392 belongs to the family Myoviridae. The host range, the multiplicity of infection, and a one-step growth curve were also determined.

Genomic analysis of bacteriophage Xoo-sp13 infecting Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae is a bacterial pathogen that gives rise to diseases in rice all over the world. A bacteriophage infecting this bacterium was isolated from rice fields in China. Here, we report the complete genome sequence of this phage, which has a linear dsDNA genome of 309,023 bp and a G + C content of 42.43%. It contains 401 open reading frames and encodes 28 tRNAs. It belongs to the family Myoviridae and has a broad host range, making it a possible candidate for phage therapy.

Isolation, characterization and genomic analysis of vB-AhyM-AP1, a lytic bacteriophage infecting Aeromonas hydrophila.

AIMS: Aeromonas hydrophila is a zoonotic pathogen displaying resistance to multiple antibiotics. Here, we aim to develop a candidate biocontrol agent against A. hydrophila. METHODS AND RESULTS: In this study, we isolated and characterized the phage vB-AhyM-AP1 from sewage. It showed lytic activity against A. hydrophila strains. One-step growth curve revealed that the latent period lasted for 40 min. The burst size of one lytic cycle was 1413 PFU per infected cell. Temperature stability studies showed that the phage vB-AhyM-AP1 was active over temperatures ranging from 4 to 45°C for 1 h. pH stability studies indicated that the phage remained active within a pH range of 5-10 after 24 h of incubation. Stability tests in salt solutions showed that the phage was stable at salinities ranging from 0·1 to 2%. The phage also showed stabilities in organic solvents when incubated for 10 min. The Illumina Hiseq sequencing of its genome indicated that the phage vB-AhyM-AP1was a jumbo phage with a genome size of 2, 54 490 bp and GC content of 40·3%. The phylogenetic analysis of the terminase large subunit and major capsid protein indicated that the phage closely clustered with other Tevenvirinae phages. The genome encoded 455 ORFs and 22 tRNAs. The phage resulted in a reduction of 0·8 log units of viable A. hydrophila cells in biofilms grown on PVC coupons maintained in a low nutrient medium for 10 days. CONCLUSIONS: The phage showed lytic activity against planktonic and biofilm cells of A. hydrophila. Genome-based prediction showed it to be a strictly lytic phage without any virulence or antibiotic resistance genes indicating safety for environmental and clinical applications. SIGNIFICANCE AND IMPACT OF THE STUDY: The multidrug-resistant strains of A. hydrophila pose a significant health risk to both cultured fishes and consumers leaving few options for treatment. Phage vB-AhyM-AP1 may be used as a candidate biocontrol agent against A. hydrophila strains.

Characterization and Genome Study of Novel Lytic Bacteriophages against Prevailing Saprophytic Bacterial Microflora of Minimally Processed Plant-Based Food Products.

The food industry is still searching for novel solutions to effectively ensure the microbiological safety of food, especially fresh and minimally processed food products. Nowadays, the use of bacteriophages as potential biological control agents in microbiological food safety and preservation is a promising strategy. The aim of the study was the isolation and comprehensive characterization of novel bacteriophages with lytic activity against saprophytic bacterial microflora of minimally processed plant-based food products, such as mixed leaf salads. From 43 phages isolated from municipal sewage, four phages, namely Enterobacter phage KKP 3263, Citrobacter phage KKP 3664, Enterobacter phage KKP 3262, and Serratia phage KKP 3264 have lytic activity against Enterobacter ludwigii KKP 3083, Citrobacter freundii KKP 3655, Enterobacter cloacae KKP 3082, and Serratia fonticola KKP 3084 bacterial strains, respectively. Transmission electron microscopy (TEM) and whole-genome sequencing (WGS) identified Enterobacter phage KKP 3263 as an Autographiviridae, and Citrobacter phage KKP 3664, Enterobacter phage KKP 3262, and Serratia phage KKP 3264 as members of the Myoviridae family. Genome sequencing revealed that these phages have linear double-stranded DNA (dsDNA) with sizes of 39,418 bp (KKP 3263), 61,608 bp (KKP 3664), 84,075 bp (KKP 3262), and 148,182 bp (KKP 3264). No antibiotic resistance genes, virulence factors, integrase, recombinase, or repressors, which are the main markers of lysogenic viruses, were annotated in phage genomes. Serratia phage KKP 3264 showed the greatest growth inhibition of Serratia fonticola KKP 3084 strain. The use of MOI 1.0 caused an almost 5-fold decrease in the value of the specific growth rate coefficient. The phages retained their lytic activity in a wide range of temperatures (from -20 °C to 50 °C) and active acidity values (pH from 4 to 11). All phages retained at least 70% of lytic activity at 60 °C. At 80 °C, no lytic activity against tested bacterial strains was observed. Serratia phage KKP 3264 was the most resistant to chemical factors, by maintaining high lytic activity across a broader range of pH from 3 to 11. The results indicated that these phages could be a potential biological control agent against saprophytic bacterial microflora of minimally processed plant-based food products.

Computational image analysis of the baseplate-tail complex of O1 ElTor vibriophage M4.

We performed an in-depth computational image analysis of the baseplate-tail complex of the M4 vibriophage and identified seven major densities in its baseplate, which notably share structural similarities with baseplate modules of a number of other bacteriophages belonging to different species. Employing computational analysis, we explained the helical organization of the sheath protein, wrapping the tail tube. Based on the results obtained in this work along with the proteomics information published previously, we are able to decipher the plausible roles assigned to the different components of the M4 baseplate during infection of the host.

Isolation and complete genome sequence of a novel cyanophage, S-B05, infecting an estuarine Synechococcus strain: insights into environmental adaptation.

A new cyanophage, S-B05, infecting a phycoerythrin-enriched (PE-type) Synechococcus strain was isolated by the liquid infection method, and its morphology and genetic features were examined. Phylogenetic analysis and morphological observation confirmed that S-B05 belongs to the family Myoviridae of the order Caudovirales. Its genome was fully sequenced, and found to be 208,857 bp in length with a G + C content of 39.9%. It contained 280 potential open reading frames and 123 conserved domains. Ninety-eight functional genes responsible for cyanophage structuring and packaging, DNA replication and regulation, and photosynthesis were identified, as well as genes encoding 172 hypothetical proteins. The genome of S-B05 is most similar to that of Prochlorococcus phage P-TIM68. Homologues of open reading frames of S-B05 can be found in various marine environments, as revealed by comparison of the S-B05 genome sequence to sequences in marine viral metagenomic databases. The presence of auxiliary metabolic genes (AMGs) related to photosynthesis, carbon metabolism, and phosphorus assimilation, as well as the phylogenetic relationships based on AMGs and the complete genome sequence, reflect the phage-host interaction mechanism or the specific adaptation strategy of the host to environmental conditions. The genome sequence information reported here will provide an important basis for further study of the adaptive evolution and ecological role of cyanophages and their hosts in the marine environment.

Complete genome analysis of Pantoea agglomerans-infecting bacteriophage vB_PagM_AAM22.

A novel myovirus, vB_PagM_AAM22 (AAM22), was isolated in Lithuania using Pantoea agglomerans as the host for phage propagation. The 49,744-bp genome of AAM22 has a G + C content of 48.4% and contains 96 probable protein-encoding genes and no genes for tRNA. In total, 34 ORFs were given a putative functional annotation, including genes associated with virion morphogenesis, DNA metabolism, and phage-host interactions. Based on comparative phylogenetic analysis, AAM22 cannot be assigned to any genus currently recognized by the ICTV and is a potential candidate to form a new genus within the family Myoviridae.

Phage vB_BveM-Goe7 represents a new genus in the subfamily Bastillevirinae.

Bacillus velezensis FZB42 is a Gram-positive, endospore-forming rhizobacterium that is associated with plant roots and promotes plant growth. It was used as host to isolate phage vB_BveM-Goe7 (Goe7). Goe7 exhibits a Myoviridae morphology with a contractile tail and an icosahedral head. Its genome is 158,674 bp in size and contains 5137-bp-long terminal repeats (LTRs). It also contains five tRNA-encoding genes and 251 coding DNA sequences (CDS), of which 65 were annotated. The adsorption constant of Goe7 is 6.1 ± 0.24 × 10-8 ml/min, with a latency period of 75 min and a burst size of 114 particles per burst. A BLASTn sequence comparison against the non-redundant nucleotide database of NCBI revealed that Goe7 is most similar to Bacillus subtilis phage vB_BsuM-Goe3.

Complete genome sequence of Buttiauxella phage vB_ButM_GuL6.

We present here the results of the analysis of the complete genome sequence of a lytic bacteriophage, vB_ButM_GuL6, which is the first virus isolated from Buttiauxella. Electron microscopy revealed that vB_ButM_GuL6 belongs to the family Myoviridae, order Caudovirales. The genome of vB_ButM_GuL6 is a linear, circularly permuted 178,039-bp dsDNA molecule with a GC content of 43.4%. It has been predicted to contain 282 protein-coding genes and two tRNA genes, tRNA-Met and tRNA-Gly. Using bioinformatics approaches, 99 (36%) of the vB_ButM_GuL6 genes were assigned a putative function. Genome-wide comparisons and phylogenetic analysis indicated that vB_ButM_GuL6 represents a new species of the subfamily Tevenvirinae and is most closely related to Escherichia virus RB43. These phages, together with Cronobacter phages Miller, CfP1, and IME-CF2, likely form a new genus within the subfamily Tevenvirinae.

Complete genome analysis of PaGz-1 and PaZq-1, two novel phages belonging to the genus Pakpunavirus.

Pseudomonas phages PaGz-1 and PaZq-1, two new phages infecting Pseudomonas aeruginosa, were isolated from fresh water in Guangdong province, China. The genomes of these two phages consist of 93,975 bp and 94,315 bp and contain 175 and 172 open reading frames (ORFs), respectively. The genome sequences of PaGz-1 and PaZq-1 share 95.8% identity with a query coverage of 94%, suggesting that these two phages belong to two different species. Based on results of nucleotide sequence alignment, gene annotation, and phylogenetic analysis, we propose PaGz-1 and PaZq-1 as representative isolates of two species in the genus Pakpunavirus within the family Myoviridae.

Characterization and genome sequencing of three Aeromonas hydrophila-specific phages, CF8, PS1, and PS2.

Aeromonas hydrophila is an important finfish pathogen, besides being an opportunistic human pathogen. In the present study, the genomes of three A. hydrophila-specific phages, CF8, PS1, and PS2, were isolated, characterized and sequenced. Transmission electron microscopy showed that all three phages had typical Myoviridae morphology. The linear dsDNA genomes of CF8, PS1, and PS2 were 238,150 bp, 237,367 bp, and 240,447 bp in length, with a GC content of 42.2%, 38.8%, and 38.8%, respectively. The low sequence similarity (67.6% - 69.8% identity with 27.0% - 29.0% query coverage) to other phage genomes in the NCBI database indicated the novel nature of the CF8, PS1, and PS2 genomes. A total of 244, 247, and 250 open reading frames (ORFs) were predicted in the CF8, PS1, and PS2 genome, respectively. During the annotation process, functional predictions were made for 28-31 ORFs, while the rest were classified as "hypothetical proteins" with yet unknown functions. Genes for tRNAs were also detected in all phage genomes. As all three phages in the present study had a very narrow host range with lytic activity against only one strain of A. hydrophila, these phages could be good candidates for phage typing applications. Moreover, the endolysin- and lytic-transglycosylase-encoding genes could be used for recombinant cloning and expression of anti-microbial proteins.

Phage vB_BmeM-Goe8 infecting Bacillus megaterium DSM319.

vB_BmeM-Goe8 is a phage preying on Bacillus megaterium. Its genome has a GC content of 38.9%, is 161,583 bp in size, and has defined ends consisting of 7436-bp-long terminal repeats. It harbours 11 genes encoding tRNAs and 246 coding DNA sequences, 66 of which were annotated. The particle reveals Myoviridae morphology, and the formation of a double baseplate upon tail sheath contraction indicates morphological relatedness to the group of SPO1-like phages. BLASTn comparison against the NCBI non-redundant nucleotide database revealed that Bacillus phage Mater is the closest relative of vB_BmeM-Goe8.

Susceptibility of Pseudomonas aeruginosa veterinary isolates to Pbunavirus PB1-like phages.

In recent years, antimicrobial-resistant Pseudomonas aeruginosa strains have increased in the veterinary field. Therefore, phage therapy has received significant attention as an approach for overcoming antimicrobial resistance. In this context, we isolated and characterized four Pseudomonas bacteriophages. Phylogenetic analysis showed that the isolated phages are novel Myoviridae Pbunavirus PB1-like phages with ØR12 belonging to a different clade compared with the other three. These phages had distinct lytic activity against 22 P. aeruginosa veterinary isolates. The phage cocktail composed from the PB1-like phages clearly inhibited the occurrence of the phage-resistant variant, suggesting that these phages could be useful in phage therapy.

Isolation, Characterisation and Experimental Evolution of Phage that Infect the Horse Chestnut Tree Pathogen, Pseudomonas syringae pv. aesculi.

Bleeding canker of horse chestnut trees is a bacterial disease, caused by the bacterium Pseudomonas syringae pv. aesculi, estimated to be present in ~ 50% of UK horse chestnut trees. Currently, the disease has no cure and tree removal can be a common method of reducing inoculum and preventing spread. One potential method of control could be achieved using naturally occurring bacteriophages infective to the causative bacterium. Bacteriophages were isolated from symptomatic and asymptomatic horse chestnut trees in three locations in the South East of England. The phages were found to be belonging to both the Myoviridae and Podoviridae families by RAPD PCR and transmission electron microscopy. Experimental coevolution was carried out to understand the dynamics of bacterial resistance and phage infection and to determine whether new infective phage genotypes would emerge. The phages exhibited different coevolution patterns with their bacterial hosts across time. This approach could be used to generate novel phages for use in biocontrol cocktails in an effort to reduce the potential emergence of bacterial resistance.