Complete genome sequences of Klebsiella pneumoniae bacteriophages YMR1 and YMR2 isolated from sewage.

Two Klebsiella pneumoniae bacteriophages, YMR1 and YMR2, which form plaques with halos, were isolated from sewage in Seoul, South Korea. YMR1 and YMR2 have double-stranded DNA genomes of 40,338 bp and 40,756 bp with 49 and 52 predicted protein-coding genes, respectively. Both are predicted to be members of the family Autographiviridae.

Genome sequences of the first Autographiviridae phages infecting marine Roseobacter.

The ubiquitous and abundant marine phages play critical roles in shaping the composition and function of bacterial communities, impacting biogeochemical cycling in marine ecosystems. Autographiviridae is among the most abundant and ubiquitous phage families in the ocean. However, studies on the diversity and ecology of Autographiviridae phages in marine environments are restricted to isolates that infect SAR11 bacteria and cyanobacteria. In this study, ten new roseophages that infect marine Roseobacter strains were isolated from coastal waters. These new roseophages have a genome size ranging from 38 917 to 42 634 bp and G+C content of 44.6-50 %. Comparative genomics showed that they are similar to known Autographiviridae phages regarding gene content and architecture, thus representing the first Autographiviridae roseophages. Phylogenomic analysis based on concatenated conserved genes showed that the ten roseophages form three distinct subgroups within the Autographiviridae, and sequence analysis revealed that they belong to eight new genera. Finally, viromic read-mapping showed that these new Autographiviridae phages are widely distributed in global oceans, mostly inhabiting polar and estuarine locations. This study has expanded the current understanding of the genomic diversity, evolution and ecology of Autographiviridae phages and roseophages. We suggest that Autographiviridae phages play important roles in the mortality and community structure of roseobacters, and have broad ecological applications.

New Genera and Species of Caulobacter and Brevundimonas Bacteriophages Provide Insights into Phage Genome Evolution.

Previous studies have identified diverse bacteriophages that infect Caulobacter vibrioides strain CB15 ranging from small RNA phages to four genera of jumbo phages. In this study, we focus on 20 bacteriophages whose genomes range from 40 to 60 kb in length. Genome comparisons indicated that these diverse phages represent six Caulobacter phage genera and one additional genus that includes both Caulobacter and Brevundimonas phages. Within species, comparisons revealed that both single base changes and inserted or deleted genetic material cause the genomes of closely related phages to diverge. Among genera, the basic gene order and the orientation of key genes were retained with most of the observed variation occurring at ends of the genomes. We hypothesize that the nucleotide sequences of the ends of these phage genomes are less important than the need to maintain the size of the genome and the stability of the corresponding mRNAs.

Whole genome sequence analysis of Aeromonas-infecting bacteriophage AHPMCC7, a new species of genus Ahphunavirus and its application in Litopenaeus vannamei culture.

Aeromonas hydrophila, a Gram-negative pathogenic bacterium, is responsible for huge economic losses in aquaculture. In this study, we evaluated the efficacy of bacteriophage AHPMCC7 which was isolated by using A. hydrophila MTCC 1739 as a host. This bacteriophage exhibited 10 min latent period and burst size was 275. In liquid culture, bacteriophage AHPMCC7 could completely lyse A. hydrophila MTCC 1739 after 2 h. AHPMCC7 genome was 42,277 bp long with 58.9% G + C content. The genome consisted of 48 CDSs and no tRNA. The comparative genomic analyses clearly implied that AHPMCC7 might represent a novel species of the genus Aphunavirus under Autographiviridae family. Bacteriophage AHPMCC7 could survive at broad pH (3-10), temperature (4-37 °C), and salinity (0-40 ppt). In aquarium trial, AHPMCC7 could control A. hydrophila MTCC 1739 without affecting the survivability of Litopenaeus vannamei. Clearly, the bacteriophage AHPMCC7 might be used in shrimp aquaculture as a biocontrol agent.

Diverse Durham collection phages demonstrate complex BREX defense responses.

Bacteriophages (phages) outnumber bacteria ten-to-one and cause infections at a rate of 1025 per second. The ability of phages to reduce bacterial populations makes them attractive alternative antibacterials for use in combating the rise in antimicrobial resistance. This effort may be hindered due to bacterial defenses such as Bacteriophage Exclusion (BREX) that have arisen from the constant evolutionary battle between bacteria and phages. For phages to be widely accepted as therapeutics in Western medicine, more must be understood about bacteria-phage interactions and the outcomes of bacterial phage defense. Here, we present the annotated genomes of 12 novel bacteriophage species isolated from water sources in Durham, UK, during undergraduate practical classes. The collection includes diverse species from across known phylogenetic groups. Comparative analyses of two novel phages from the collection suggest they may be founding members of a new genus. Using this Durham phage collection, we determined that particular BREX defense systems were likely to confer a varied degree of resistance against an invading phage. We concluded that the number of BREX target motifs encoded in the phage genome was not proportional to the degree of susceptibility. IMPORTANCE Bacteriophages have long been the source of tools for biotechnology that are in everyday use in molecular biology research laboratories worldwide. Phages make attractive new targets for the development of novel antimicrobials. While the number of phage genome depositions has increased in recent years, the expected bacteriophage diversity remains underrepresented. Here we demonstrate how undergraduates can contribute to the identification of novel phages and that a single City in England can provide ample phage diversity and the opportunity to find novel technologies. Moreover, we demonstrate that the interactions and intricacies of the interplay between bacterial phage defense systems such as Bacteriophage Exclusion (BREX) and phages are more complex than originally thought. Further work will be required in the field before the dynamic interactions between phages and bacterial defense systems are fully understood and integrated with novel phage therapies.

The metastable associations of bacteriophages and Erwinia amylovora.

Bacteriophages are often considered as possible agents of biological control of unwanted bacterial populations in medicine, agriculture and food industry. Although the virulent phages can efficiently kill the infected host cells but at the population level phage attack not always leads to the host population collapse but may result in establishment of a more or less stable co-existence. The mechanism of the long-term stabilization of the mixed phage-host cultures is poorly understood. Here we describe bacteriophages VyarbaL and Hena2, the members of the Molineuxvirinae and the Ounavirinae subfamilies, respectively, that are able to form the pseudolysogenic associations (PA) with their host Erwinia amylovora 1/79Sm on solid media. These PAs were stable through multiple passages. The phenomenon of the PA formation between a bacterial culture and bacteriophages decreases the effectiveness of bacteriophage-mediated biological control agents based on lytic bacteriophages.

A Pseudomonas Lysogenic Bacteriophage Crossing the Antarctic and Arctic, Representing a New Genus of Autographiviridae.

Polar regions tend to support simple food webs, which are vulnerable to phage-induced gene transfer or microbial death. To further investigate phage-host interactions in polar regions and the potential linkage of phage communities between the two poles, we induced the release of a lysogenic phage, vB_PaeM-G11, from Pseudomonas sp. D3 isolated from the Antarctic, which formed clear phage plaques on the lawn of Pseudomonas sp. G11 isolated from the Arctic. From permafrost metagenomic data of the Arctic tundra, we found the genome with high-similarity to that of vB_PaeM-G11, demonstrating that vB_PaeM-G11 may have a distribution in both the Antarctic and Arctic. Phylogenetic analysis indicated that vB_PaeM-G11 is homologous to five uncultured viruses, and that they may represent a new genus in the Autographiviridae family, named Fildesvirus here. vB_PaeM-G11 was stable in a temperature range (4-40 °C) and pH (4-11), with latent and rise periods of about 40 and 10 min, respectively. This study is the first isolation and characterization study of a Pseudomonas phage distributed in both the Antarctic and Arctic, identifying its lysogenic host and lysis host, and thus provides essential information for further understanding the interaction between polar phages and their hosts and the ecological functions of phages in polar regions.

Three Phages One Host: Isolation and Characterization of Pantoea agglomerans Phages from a Grasshopper Specimen.

The bacterial genus Pantoea comprises species found in a variety of different environmental sources. Pantoea spp. are often recovered from plant material and are capable of both benefitting the plants and acting like phytopathogens. Some species of Pantoea (including P. agglomerans) are considered opportunistic human pathogens capable of causing various infections in immunocompromised subjects. In this study, a strain of P. agglomerans (identified by 16S rRNA gene sequencing) was isolated from a dead specimen of an unidentified Latvian grasshopper species. The retrieved strain of P. agglomerans was then used as a host for the potential retrieval of phages from the same source material. After rounds of plaque purification and propagation, three high-titer lysates corresponding to putatively distinct phages were acquired. Transmission electron microscopy revealed that one of the phages was a myophage with an unusual morphology, while the two others were typical podophages. Whole-genome sequencing (WGS) was performed for each of these isolated phages. Genome de novo assembly and subsequent functional annotation confirmed that three different strictly lytic phages were isolated. Elaborate genomic characterization of the acquired phages was performed to elucidate their place within the so-far-uncovered phage diversity.

Isolation and genomic analysis of a novel bacteriophage IME278 infecting Enterobacter hormaechei and its biocontrol potential on pork.

Enterobacter hormaechei is an opportunistic pathogen and is found in a large variety of food including animal-derived food. In recent years, bacteria present a severe clinical challenge due to their increasing resistance to antibiotics. Bacteriophages have gained attention as a new antibacterial strategy. In this study, we isolated a novel E. hormaechei bacteriophage IME278 from hospital sewage in Beijing, China. Bacteriophage IME278 had a double-stranded linear DNA genome with 40,164 bp and 51.99% GC content. Whole-genome alignments showed IME278 shared 87% homology with other phages in the National Center for Biotechnology Information (NCBI) database. And phylogenetic analysis demonstrated that IME278 was highly similar to bacteriophages belonging to the genus Kayfunavirus, family Autographiviridae, indicating IME278 can be classified as a new member of the Autographiviridae family. Transmission electron microscopy revealed that IME278 had an icosahedral head 51.72 nm in diameter and a tail 151.28 nm in length. Bacteriophage IME278 was able to survive under high temperature (50 °C-70 °C) and its activity decreased significantly above 70 °C and almost completely inactivated at 80 °C. Bacteriophage IME278 could survive in a wide pH range (4.0-11.0) and it was stable in chloroform (up to 5%). The phage was sensitive to UV irradiation. Bacteriophage IME278 had a latent period of 40 min and reached a plateau stage at 150 min and its cleavage was approximately 8.21 × 108/3.66 × 108 = 2.24. The biocontrol potential of bacteriophage IME278 was evaluated in a model that artificially contaminated pork with E. hormaechei 529 and the result revealed that IME278 could effectively control bacterial contamination on pork. The in-depth analysis of the biological characteristics, whole genome sequencing, and bioinformatics of IME278 has laid the foundation for the biocontrol application and the treatment of bacteria using bacteriophages.

StenM_174: A Novel Podophage That Infects a Wide Range of Stenotrophomonas spp. and Suggests a New Subfamily in the Family Autographiviridae.

Stenotrophomonas maltophilia was discovered as a soil bacterium associated with the rhizosphere. Later, S. maltophilia was found to be a multidrug-resistant hospital-associated pathogen. Lytic bacteriophages are prospective antimicrobials; therefore, there is a need for the isolation and characterization of new Stenotrophomonas phages. The phage StenM_174 was isolated from litter at a poultry farm using a clinical strain of S. maltophilia as the host. StenM_174 reproduced in a wide range of clinical and environmental strains of Stenotrophomonas, mainly S. maltophilia, and it had a podovirus morphotype. The length of the genomic sequence of StenM_174 was 42,956 bp, and it contained 52 putative genes. All genes were unidirectional, and 31 of them encoded proteins with predicted functions, while the remaining 21 were identified as hypothetical ones. Two tail spike proteins of StenM_174 were predicted using AlphaFold2 structural modeling. A comparative analysis of the genome shows that the Stenotrophomonas phage StenM_174, along with the phages Ponderosa, Pepon, Ptah, and TS-10, can be members of the new putative genus Ponderosavirus in the Autographiviridae family. In addition, the analyzed data suggest a new subfamily within this family.

A Novel Aeromonas popoffii Phage AerP_220 Proposed to Be a Member of a New Tolavirus Genus in the Autographiviridae Family.

Aeromonas popoffii is one of the environmental Aeromonas species. A number of factors of virulence have been described for this species and it has been reported as a causative agent of urinary tract infection. The first A. popoffii bacteriophage AerP_220 along with its host strain A. popoffii CEMTC 4062 were isolated from river water. The phage has a podovirus morphotype, shows a narrow host range and is lytic against the host strain. The AerP_220 genome comprises 45,207 bp and does not contain genes responsible for antibiotic resistance and toxin production. Fifty-nine co-directional putative ORFs were found in the AerP_220 genome. Thirty-three ORFs encoded proteins with predicted functions; the products of 26 ORFs were hypothetical proteins. AerP_220 genome analysis revealed that this phage can be considered a novel species within the Autographiviridae family. Comparative genomic and proteomic analysis revealed that AerP_220 along with the Aeromonas phage vB_AspA_Tola (OM913599) are members of a new putative Tolavirus genus in the family Autographiviridae. The Gajwadongvirus and proposed Tolavirus genera along with Pantoea phage Nufs112 and phage Reminis could form a new Tolavirinae subfamily within the Autographiviridae family.

Characterization and Genomic Analysis of Novel Vibrio parahaemolyticus Phage vB_VpaP_DE10.

In the present study, a novel lytic Vibrio parahaemolyticus phage, vB_VpaP_DE10, was isolated from sewage samples collected in Guangzhou city, China. Transmission electron microscopy revealed that phage vB_VpaP_DE10 has an icosahedral head (52.4 ± 2.5 nm) and a short non-contracted tail (21.9 ± 1.0 nm). Phage vB_VpaP_DE10 lysed approximately 31% (8/26) of the antibiotic-resistant V. parahaemolyticus strains tested. A one-step growth curve showed that phage vB_VpaP_DE10 has a relatively long latency time of 25 min and a burst size of ~19 PFU per cell. The genome of phage vB_VpaP_DE10 is a 42,871-bp-long dsDNA molecule with a G + C content of 49.19% and is predicted to contain 46 open reading frames, 26 of which are predicted to be related to functions such as phage structure, packaging, host lysis, and DNA metabolism. Sequence comparisons suggested that vB_VpaP_DE10 is a member of the genus Maculvirus within the family Autographiviridae. Morphological and genomic analysis indicated that vB_VpaP_DE10 is a novel V. parahaemolyticus phage.

Isolation and genomic characterization of a novel Autographiviridae bacteriophage IME184 with lytic activity against Klebsiella pneumoniae.

Klebsiella pneumoniae, a multidrug resistant bacterium that causes nosocomial infections including septicemia, pneumonia etc. Bacteriophages are potential antimicrobial agents for the treatment of antibiotic resistant bacteria. In this study, a novel bacteriophage IME184, was isolated from hospital sewage against clinical multi-drug resistant Klebsiella pneumoniae. Transmission electron microscopy and genomic characterization exhibited this phage belongs to the Molineuxvirinae genus, Autographiviridae family. Phage IME184 possessed a double-stranded DNA genome composed of 44,598 bp with a GC content of 50.3%. The phage genome encodes 57 open reading frames, out of 26 are hypothetical proteins while 31 had assigned putative functions. No tRNA, virulence related or antibiotic resistance genes were found in phage genome. Comparative genomic analysis showed that phage IME184 has 94% similarity with genomic sequence of Klebsiella phage K1-ULIP33 (MK380014.1). Multiplicity of infection, one step growth curve and host range of phage were also measured. According to findings, Phage IME184 is a promising biological agent that infects Klebsiella pneumoniae and can be used in future phage therapies.

Characterization of Three Novel Virulent Aeromonas Phages Provides Insights into the Diversity of the Autographiviridae Family.

In this study, we isolated and characterized three novel virulent Autographiviridae bacteriophages, vB_AspA_Bolek, vB_AspA_Lolek, and vB_AspA_Tola, which infect different Aeromonas strains. These three host-pathogen pairs were derived from the same sampling location-the arsenic-containing microbial mats of the Zloty Stok gold mine. Functional analysis showed they are psychrotolerant (4-25 °C), albeit with a much wider temperature range of propagation for the hosts (≤37 °C). Comparative genomic analyses revealed a high nucleotide and amino acid sequence similarity of vB_AspA_Bolek and vB_AspA_Lolek, with significant differences exclusively in the C-terminal region of their tail fibers, which might explain their host range discrimination. The protein-based phage network, together with a phylogenetic analysis of the marker proteins, allowed us to assign vB_AspA_Bolek and vB_AspA_Lolek to the Beijerinckvirinae and vB_AspA_Tola to the Colwellvirinae subfamilies, but as three novel species, due to their low nucleotide sequence coverage and identity with other known phage genomes. Global comparative analysis showed that the studied phages are also markedly different from most of the 24 Aeromonas autographiviruses known so far. Finally, this study provides in-depth insight into the diversity of the Autographiviridae phages and reveals genomic similarities between selected groups of this family as well as between autographiviruses and their relatives of other Caudoviricetes families.

Isolation and Characterization of Two Lytic Phages Efficient Against Phytopathogenic Bacteria From Pseudomonas and Xanthomonas Genera.

Pseudomonas syringae is a bacterial pathogen that causes yield losses in various economically important plant species. At the same time, P. syringae pv. tomato (Pst) is one of the best-studied bacterial phytopathogens and a popular model organism. In this study, we report on the isolation of two phages from the market-bought pepper fruit showing symptoms of bacterial speck. These Pseudomonas phages were named Eir4 and Eisa9 and characterized using traditional microbiological methods and whole-genome sequencing followed by various bioinformatics approaches. Both of the isolated phages were capable only of the lytic life cycle and were efficient against several pathovars from Pseudomonas and Xanthomonas genera. With the combination of transmission electron microscopy (TEM) virion morphology inspection and comparative genomics analyses, both of the phages were classified as members of the Autographiviridae family with different degrees of novelty within the known phage diversity. Eir4, but not Eisa9, phage application significantly decreased the propagation of Pst in the leaf tissues of Arabidopsis thaliana plants. The biological properties of Eir4 phage allow us to propose it as a potential biocontrol agent for use in the prevention of Pst-associated bacterioses and also as a model organism for the future research of mechanisms of phage-host interactions in different plant systems.

Genomic and biological characteristics of a newly isolated lytic bacteriophage PZJ0206 infecting the Enterobacter cloacae.

Enterobacter cloacae is an opportunistic pathogen that widely distributes in various environments, often causing hospital and community-acquired infections. The limited phage resources and poor genomic data hinder the understanding of the lysis mechanism of Enterobacter cloacae phages. In the present study, we characterized a newly discovered lytic phage PZJ0206 that infects an E. cloacae strain isolated from sewage water into the Pearl River in Guangdong, China. The morphological characteristics of the icosahedral head and short tail of PZJ0206 were observed by transmission electron microscopy. The one-step growth curve showed that PZJ0206 has a relatively short latent period and an average burst size of approximately 124 PFU (plaque forming unit)/infected cell at an MOI of 0.1. The genome of phage PZJ0206 consists of 40,526 bp with a GC content of 48.62% and contains 45 putative open reading frames (ORFs). Based on genomic characteristics, comparative genomics and phylogenetic analysis, it was strongly demonstrated that PZJ0206 is a novel member of the genus Berlinvirus in the family Autographiviridae. Our work provides basic information on Enterobacter cloacae phage PZJ0206 and lays the foundation for the further study of the evolution of phage PZJ0206 and interactions between the phage and its host.

Identification and Characterization of vB_PreP_EPr2, a Lytic Bacteriophage of Pan-Drug Resistant Providencia rettgeri.

Providencia rettgeri is an emerging opportunistic Gram-negative pathogen with reports of increasing antibiotic resistance. Pan-drug resistant (PDR) P. rettgeri infections are a growing concern, demonstrating a need for the development of alternative treatment options which is fueling a renewed interest in bacteriophage (phage) therapy. Here, we identify and characterize phage vB_PreP_EPr2 (EPr2) with lytic activity against PDR P. rettgeri MRSN 845308, a clinical isolate that carries multiple antibiotic resistance genes. EPr2 was isolated from an environmental water sample and belongs to the family Autographiviridae, subfamily Studiervirinae and genus Kayfunavirus, with a genome size of 41,261 base pairs. Additional phenotypic characterization showed an optimal MOI of 1 and a burst size of 12.3 ± 3.4 PFU per bacterium. EPr2 was determined to have a narrow host range against a panel of clinical P. rettgeri strains. Despite this fact, EPr2 is a promising lytic phage with potential for use as an alternative therapeutic for treatment of PDR P. rettgeri infections.

Sheep in wolves' clothing: Temperate T7-like bacteriophages and the origins of the Autographiviridae.

Bacteriophage T7 is an extensively studied virulent phage, and its taxonomic family, the Autographiviridae, is broadly synonymous with a strictly virulent lifestyle. It is difficult to imagine how a T7-like phage could function in a "domesticated" temperate lifestyle, in which it is incorporated into the host's genome. Here we describe two temperate T7-like bacteriophages: ProddE, a Desulfovibrio phage, and Pasto, an Agrobacterium phage. Each contains recognizable T7-like proteins in the canonical T7-like gene order, but with the addition of lysogeny gene modules. While ProddE contains a phage-like repressor, Pasto lysogeny appears to be controlled by a novel MarR-like transcriptional regulator. In addition, we identify similar T7-like prophage elements in a wide variety of Gram-negative bacterial genomes and a small number of Gram-positive genomes. Identification of these elements in diverse bacterial species raises interesting evolutionary questions about the origins of T7-like phages and which lifestyle, temperate or virulent, is the ancestral form.

Isolation and Characterization of a Novel Autographiviridae Phage and Its Combined Effect with Tigecycline in Controlling Multidrug-Resistant Acinetobacter baumannii-Associated Skin and Soft Tissue Infections.

Multidrug-resistant Acinetobacter baumannii (MDR A. baumannii) is one of the ESKAPE pathogens that restricts available treatment options. MDR A. baumannii is responsible for a dramatic increase in case numbers of a wide variety of infections, including skin and soft tissue infections (SSTIs), resulting in pyoderma, surgical debridement, and necrotizing fasciitis. To investigate an alternative medical treatment for SSTIs, a broad range lytic Acinetobacter phage, vB _AbP_ABWU2101 (phage vABWU2101), for lysing MDR A. baumannii in associated SSTIs was isolated and the biological aspects of this phage were investigated. Morphological characterization and genomic analysis revealed that phage vABWU2101 was a new species in the Friunavirus, Beijerinckvirinae, family Autographiviridae, and order Caudovirales. Antibiofilm activity of phage vABWU2101 demonstrated good activity against both preformed biofilms and biofilm formation. The combination of phage vABWU2101 and tigecycline showed synergistic antimicrobial activities against planktonic and biofilm cells. Scanning electron microscopy confirmed that the antibacterial efficacy of the combination of phage vABWU2101 and tigecycline was more effective than the phage or antibiotic alone. Hence, our findings could potentially be used to develop a therapeutic option for the treatment of SSTIs caused by MDR A. baumannii.

Diverse Bacteriophages Infecting the Bacterial Striped Catfish Pathogen Edwardsiella ictaluri.

Bacteriophages infecting Edwardsiella ictaluri have been less investigated, although the host bacterium is one of the most important fish pathogens causing enteric septicemia of catfish (ESC). We present here two distinctly novel bacteriophages vB_EiM_PVN06 and vB_EiA_PVN09 infecting Edwardsiella ictaluri E1, with their geographical origins from the Mekong Delta, Vietnam. Bacteriophage vB_EiM_PVN06 native to a mud sample reveals complete differences of biological properties with the phage vB_EiA_PVN09 originated from a viscus of a healthy catfish (Pangasianodon hypophthalmus) cultured in the same area. Morphological analyses combined with genomic data indicate that phage vB_EiM_PVN06 is classified to Myoviridae family and shares high similarity with E. ictaluri phage PEi21 genome, while vB_EiA_PVN09 is a member of Teseptimavirus genus, Autographiviridae family, and mostly closes to phage vB_EcoP_IME390. The vB_EiA_PVN09 is a T7-like bacteriophage, which has been firstly found infecting to E. ictaluri, and host range analysis also evidences for the cross-infection of this phage to Escherichia coli K12 and Escherichia coli DH5α. Together, our research highlights the diversity of bacteriophages infecting the pathogen E. ictaluri and suggests further explorations of lytic phages in environmental niches, to be exploited in feasible strategies of phage therapy in ESC disease control.