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Salmonella is a significant food-borne pathogen that infects a large number of people worldwide. In this study, a lytic bacteriophage vB_SenS_SE1 capable of infecting Salmonella is isolated from municipal wastewater in Beijing, and its biological and genomic features are analyzed. Transmission electron micrograph shows that vB_SenS_SE1 is likely a Siphoviridae virus, with an icosahedral head and a long non-contracted tail. The stability test in vitro reveals that it is stable at 4-50 °C and pH 4-12. Based on the one-step growth curve, vB_SenS_SE1 has a 60-min exponential phase and a low burst size (19 PFU per cell). Bioinformatics analysis reveals that vB_SenS_SE1 consists of a circular, double-stranded DNA molecule of 40,987 bp with a GC content of 51.2%. Its genome carries 63 predicted open reading frames (orfs), with 22 orfs encoding known proteins. Phylogenetic analysis of the large terminase subunit shows that vB_SenS_SE1 exhibits strong homology to Salmonella phage St161, St162, VSiP, and FSL SP-031. The CoreGenes analysis shows that it is a member of the virus genus Cornellvirus. The features of phage vB_SenS_SE1 suggest that it has the potential to be an agent to control Salmonella.

Chicken breast meat is considered as the main source of Salmonella infection in humans. The aim of this study was to isolate lytic bacteriophages specific for Salmonella enterica serovars Enteritidis and examine their efficacy in a cocktail for the biocontrol of Salmonella spp. in raw chicken breast meat. Four lytic phages belonging to the Myoviridae and Siphoviridae families were isolated from a river proximate to a duck farm. They exhibited broad lytic activities against 11 strains of S. Enteritidis, 11 strains of S. Typhimurium, and one each of S. Paratyphi A, S. San Diego, and S. Typhi. The phages were determined to be stable, exhibited similar degrees of resistance to heat and pH, and had latent periods ranging from 5 to 30 min. In addition, the phage particles were 100% adsorbed within 18 to 40 min. Viable cell counts of bacteria were significantly reduced in raw chicken breast samples (P < 0.05) when treated with a cocktail of all four bacteriophages at 4 °C for 7 days (multiplicities of infection were from 104 to 106 ). These results indicate the potential efficacy of the bacteriophage cocktail as a biological agent against S. Enteritidis in raw chicken breast meat. PRACTICAL APPLICATION: Our findings demonstrate that the phages could be effective in reducing the viability of Salmonella spp. bacteria in chicken breast meat. Therefore, the phage cocktail is a potential bactericidal agent for the biocontrol of Salmonella spp. in raw chicken breast meat and could be used use in various poultry industries in the future.

The long flexible tail tube of bacteriophage lambda connects its capsid to the tail tip. On infection, a DNA ejection signal is passed from the tip, along the tube to the capsid that triggers passage of the DNA down the tube and into the host bacterium. The tail tube is built from repeating units of the major tail protein, gpV, which has two distinctive domains. Its N-terminal domain has the same fold as proteins that form the rigid inner tubes of contractile tail phages, such as T4, and its C-terminal domain adopt an Ig-like fold of unknown function. We determined structures of the lambda tail tube in free tails and in virions before and after DNA ejection using cryoelectron microscopy. Modeling of the density maps reveals how electrostatic interactions and a mobile loop participate in assembly and also impart flexibility to the tube while maintaining its integrity. We also demonstrate how a common protein fold produces rigid tubes in some phages but flexible tubes in others.

The cyanobacteria constitute a diverse group of prokaryotic organisms widespread in aquatic ecosystems. Microcystis is a genus of freshwater cyanobacteria, which poses a possible cause for concern due to its excessive growth and potential for toxin production, which can be aggravated by intensive eutrophication of freshwaters and climate changes. Factors that can potentially affect the biology, ecology and evolution of cyanobacteria are cyanophages. This paper summarizes the current state of knowledge regarding cyanophages specific to Microcystis sp. in the natural environment of East Asia and Central Europe. Being a group of planktonic viruses, cyanophages have been studied since the 1960s; information on viruses specific to Microcystis sp. was first obtained in the 1970s and are being dynamically developing over the last two decades. There are eight strains of cyanophages capable of lysing Microcystis sp. cells reported to date, belonging to Myoviridae, Podoviridae and Siphoviridae families, as well as forming Corticovirus-like particles. Available literature data indicates that the gene encoding the sheath protein of the contractile tail (g91) is a good molecular marker for tracing Microcystis viruses from Myoviridae as a form of environmental analysis. It has been suggested that in natural ecosystems, this group of cyanophages can cause a shift in the host population from predominant ‘cyanophage infection-sensitive’ to ‘cyanophage infection-insensitive’ cells, rather than influencing the total number of Microcystis sp. It was found that nucleotide sequence of the g91 gene fragment isolated from Central Europe was 95% similar to that of its counterparts from East Asia. However, the research based on molecular primers specific to the Microcystis sp. viruses is limitted to two of the known strains to date. It is suggested that further studies in molecular biology and ecology of Microcystis sp. cyanophages should be based on metagenomics accompanied with physicochemical environmental variables.

A previously isolated a bacteriophage, vB_EcoS_AKFV33 of T5virus, demonstrated great potential in biocontrol of Shiga toxigenic Escherichia coli (STEC) O157. This study further evaluated its potential as a biocontrol agent in broth culture against other important non-O157 serogroups of STEC and Salmonella. AKFV33 was capable of lysing isolates of STEC serogroups O26 (n = 1), O145 (n = 1) and Salmonella enterica serovars (n = 6). In a broth culture microplate system, efficacy of AKFV33 for killing STEC O26:H11, O145:NM and Salmonella was improved (P Salmonella in mixtures with enhanced activity (P < 0.05) against O157:H7 and O26:H11, offering great promise for control of multiple zoonotic pathogens at both pre and post-harvest.

We have developed an efficient protocol using our two-layer Molecules-in-Molecules (MIM2) fragmentation-based quantum chemical method for the prediction of NMR chemical shifts of large biomolecules. To investigate the performance of our fragmentation approach and demonstrate its applicability, MIM-NMR calculations are first calibrated on a test set of six proteins. The MIM2-NMR method yields a mean absolute deviation (MAD) from unfragmented full molecule calculations of 0.01 ppm for 1H and 0.06 ppm for 13C chemical shifts. Thus, the errors from fragmentation are only about 3% of our target accuracy of ∼0.3 ppm for 1H and 2-3 ppm for 13C chemical shifts. To compare with experimental chemical shifts, a standard protocol is first derived using two smaller proteins 2LHY (176 atoms) and 2LI1 (146 atoms) for obtaining an appropriate protein structure for NMR chemical shift calculations. The effect of the solvent environment on the calculated NMR chemical shifts is incorporated through implicit, explicit, or explicit-implicit solvation models. The expensive first solvation shell calculations are replaced by a micro-solvation model in which only the immediate interaction between the protein and the explicit solvation environment is considered. A single explicit water molecule for each amine and amide proton is found to be sufficient to yield accurate results for 1H chemical shifts. The 1H and 13C NMR chemical shifts calculated using our protocol give excellent agreement with experiments for two larger proteins, 2MC5 (the helical part with 265 atoms) and 3UMK (33 residue slice with 547 atoms). Overall, our target accuracy of ∼0.3 ppm for 1H and ∼2-3 ppm for 13C has been achieved for the larger proteins. The proposed MIM-NMR method is accurate and computationally cost-effective and should be applicable to study a wide range of large proteins.

The diversity among bacteriophages depends on different factors like ecology, temperature conditions and genetic pool. Current study focused on isolation, identification and diversity of phages from 34 sewage water samples collected from two different wastewater treatment plants (WWTPs), King Saud University wastewater treatment plants (KSU-WWTP) and Manfoha wastewater treatment plants (MN-WWTP) in Riyadh, Saudi Arabia. Samples were analyzed by PCR and Next Generation Sequencing (NGS). Siphoviridae, Podoviridae and Myoviridae families were detected by family-specific PCR and highest prevalence of Myoviridae 29.40% was found at MN-WWTP followed by 11.76% at KSU-WWTP. Siphoviridae was detected 11.76% at MN-WWTP and 5.88% at KSU-WWTP. Lowest prevalence for Podoviridae family (5.88%) was recorded at MN-WWTP. Significant influence of temporal variations on prevalence of Myoviridae and Siphoviridae was detected in both WWTP and MN-WWTP, respectively. Highest phage prevalence was obtained in August (75%), followed by September (50%). Highest phage prevalence was recorded at a temperature range of 29–33°C. Significant influence of temperature on the prevalence of Myoviridae phages was detected at MN-WWTP. Four bacteriophages with various abundance levels were identified by NGS. Cronobacter virus Esp2949-1 was found first time with highest abundance (4.41%) in wastewater of Riyadh. Bordetella virus BPP1 (4.14%), Dickeya virus Limestone (1.55%) and Ralstonia virus RSA1 (1.04%) were also detected from samples of MN-WWTP. Highest occurrence of Bordetella virus BPP1 (67%) and (33.33%) was recorded at KSU-WWTP and MN-WWTP, respectively. Highest Bordetella virus BPP1 occurrence was recorded in September (50%) followed by August (40%). The findings of study showed new insights of phage diversity from wastewater sources and further large-scale data studies are suggested for comprehensive understanding.

In staphylococcal phage research, studies specific to coagulase-negative staphylococci (CoNS) remain severely under-represented, and the number of temperate bacteriophages is limited. This investigation identifies a novel temperate phage IME1354_01 from the strain Staphylococcus cohnii IME1354, which was isolated from the skin of a patient with foot ulcer disease. The phage IME1354_01 is the first isolated temperate phage of S. cohnii, and was determined to have a long-tail morphology using TEM. Its genome was found to be a 42,706-bp linear dsDNA molecule with a GC content of 34%. The integration of IME1354_01 occurred using a tRNA-Ser coding gene, and it did not affect tRNA-Ser function. The genome of IME1354_01 is most closely related to that of the temperate Staphylococcus arlettae phage vB_Sars_BM31 with 10% homology coverage and 83.73% nucleotide identity. In addition, they showed similarities mainly in the DNA replication, DNA packaging and partial morphogenesis modules. We propose that a new genus should be created for IME1354_01 based on the intergenomic similarities (maximum is 23%) obtained from the VIRIDIC calculations. The isolation and in-depth study of the novel phage, IME1354_01, will improve our understanding of the evolutionary relationship between temperate phages and their hosts.

Stenotrophomonas maltophilia is a ubiquitous environmental bacterium capable of causing disease in humans. Antibiotics are largely ineffective against this pathogen due to numerous chromosomally encoded antibiotic resistance mechanisms. An alternative treatment option is phage therapy, the use of bacteriophages to selectively kill target bacteria that are causing infection. To this aim, we isolated the Siphoviridae bacteriophage AXL1 (vB_SmaS-AXL_1) from soil and herein describe its characterization. Host range analysis on a panel of 30 clinical S. maltophilia strains reveals a moderate tropism that includes cross-species infection of Xanthomonas, with AXL1 using the type IV pilus as its host surface receptor for infection. Complete genome sequencing and analysis revealed a 63,962 bp genome encoding 83 putative proteins. Comparative genomics place AXL1 in the genus Pamexvirus, along with seven other phages that infect one of Stenotrophomonas, Pseudomonas or Xanthomonas species. Functional genomic analyses identified an AXL1-encoded dihydrofolate reductase enzyme that provides additional resistance to the antibiotic combination trimethoprim–sulfamethoxazole, the current recommended treatment option for S. maltophilia infections. This research characterizes the sixth type IV pilus-binding phage of S. maltophilia and is an example of phage-encoded antibiotic resistance.

Bacteriophages represent an enormous reservoir of novel genes, many of which are unrelated to existing entries in public databases and cannot be assigned a predicted function. Characterization of these genes can provide important insights into the intricacies of phage–host interactions and may offer new strategies to manipulate bacterial growth and behavior. Overexpression is a useful tool in the study of gene-mediated effects, and we describe here the construction of a plasmid-based overexpression library of a complete set of genes for Waterfoul, a mycobacteriophage closely related to those infecting clinically important strains of Mycobacterium tuberculosis and/or Mycobacterium abscessus. The arrayed Waterfoul gene library was systematically screened in a plate-based cytotoxicity assay, identifying a diverse set of 32 Waterfoul gene products capable of inhibiting the growth of the host Mycobacterium smegmatis and providing a first look at the frequency and distribution of cytotoxic products encoded within a single mycobacteriophage genome. Several of these Waterfoul gene products were observed to confer potent anti-mycobacterial effects, making them interesting candidates for follow-up mechanistic studies.

Here, we report the genomic characterization of two siphophages, named hairong and ZY21, that infect the kiwifruit canker phytopathogen Pseudomonas syringae pv. actinidiae. The genome sequences of hairong (112,842 bp) and ZY21 (112,006 bp) were determined. Global sequence comparison showed that hairong, ZY21, and two phages of the genus Nickievirus (nickie and psageB1) are similar but are not closely related to any other known phage, and they comprise a unique phylogenetic cluster. Moreover, hairong represents a new genus related to Nickievirus. Comparative genomic analysis revealed some common features shared by the four nickie-like phages.

Shiga-toxin-producing Escherichia coli (STEC) strains of the serogroup O157 are foodborne pathogens associated with severe clinical disease. As antibiotics are counter-indicated for treatment of these infections, they represent prime candidates for targeted application of bacteriophages to reduce infection burden. In this study, we characterised lytic bacteriophages representing three phage genera for activity against E. coli O157 strains. The phages vb_EcoM_bov9_1 (Tequatrovirus), vb_EcoM_bov11CS3 (Vequintavirus), and vb_EcoS_bov25_1D (Dhillonvirus) showed effective lysis of enterohaemorrhagic E. coli EHEC O157:H7 strains, while also exhibiting activity against other strains of the O157 serogroup, as well as of the ‘big six’ (STEC) serogroups, albeit with lower efficiency. They had a burst size of 293, 127 and 18 per cell and a latent period of 35, 5 and 30 min, respectively. In situ challenge experiments using the O157 Sakai strain on minced beef showed a reduction by 2–3-fold when treated with phages at a 0.1 MOI (multiplicity of infection), and approximately 1 log reduction when exposed to MOI values of 10 and 100. A cocktail of the phages, applied at 10 × and 100 × MOI showed 2 to 3 log reduction when samples were treated at room temperature, and all treatments at 37 °C with 100 × MOI resulted in a 5 to 6 log reduction in cell count. Our results indicate that the phages vb_EcoM_bov9_1 and vb_EcoM_bov11CS3, which have higher burst sizes, are promising candidates for biocontrol experiments aimed at the eradication of E. coli O157 strains in animals or foodstuff.

Four b acteriophage i nsensitive m utants (BIMs) of the dairy starter bacterium Streptococcus thermophilus UCCSt50 were isolated following a challenge with the Brussowvirus SW13. The BIMs displayed an altered, sedimentation phenotype. Whole genome sequencing and comparative genomic analysis of the BIMs uncovered mutations within a family-2-glycosyltransferase-encoding gene (orf06955UCCSt50) located within the variable region of the cell wall-associated r hamnose- g lucose p olymer (Rgp) biosynthesis locus (designated here as the rgp gene cluster). Complementation of a representative BIM, S. thermophilus B1, with native orf06955UCCSt50 restored phage sensitivity comparable to that of the parent strain. Detailed bioinformatic analysis of the gene product of orf06955UCCSt50 identified it as a functional homolog of the Lactococcus lactis p oly s accharide p ellicle (PSP) initiator, WpsA. Biochemical analysis of cell wall fractions of strains UCCSt50 and B1 determined that mutations within orf06955UCCSt50 result in the loss of the side chain decoration from the Rgp backbone structure. Furthermore, it was demonstrated that the intact Rgp structure incorporating the side chain structure is essential for phage binding through fluorescent labelling studies. Overall, this study confirms that the rgp gene cluster of S. thermophilus encodes the biosynthetic machinery for a cell surface-associated polysaccharide which is essential for binding and subsequent infection by Brussowviruses, thus enhancing our understanding of S. thermophilus phage-host dynamics. Importance: Streptococcus thermophilus is an important starter culture bacterium in global dairy fermentation processes, where it is used for the production of various cheeses and yogurt. Bacteriophage predation of the species can result in substandard product quality and in rare cases, complete fermentation collapse. To mitigate these risks, it is necessary to understand the phage-host interaction process which commences with the recognition of, and adsorption to, specific host-encoded cell surface receptors by bacteriophage(s). As new groups of S. thermophilus phages are being discovered, the importance of underpinning the genomic elements which specify the surface receptor(s) is apparent. Our research identifies a single gene which is critical for the biosynthesis of a saccharidic moiety required for phage adsorption to its S. thermophilus host. The acquired knowledge provides novel insights into phage-host interactions for this economically important starter species.

The emergence and worldwide spread of Methicillin-resistant Staphylococcus aureus (MRSA) pose a threat to human health. While bacteriophages are recognized as an effective alternative to treat infections caused by drug resistant pathogens, some bacteriophages in particular the temperate bacteriophage may also influence the virulence of the host bacteria in distinct ways. In this study, we isolated a bacteriophage vB_Saus_PHB21 from an epidermal sample of Siberian tiger (Panthera tigris altaica) using an MRSA strain SA14 as the indicator. Our following laboratory tests and whole genome sequencing analyses revealed that vB_Saus_PHB21 was a temperate bacteriophage belonging to the Siphoviridae family, and this bacteriophage did not contain any virulence genes. However, the integration of PHB21 genome into the host MRSA increased the bacterial capacities of cell adhesion, anti-phagocytosis, and biofilm formation. Challenge of the lysogenic strain (SA14+) caused severe mortalities in both Galleria mellonella and mouse models. Mice challenged with SA14+ showed more serious organ lesions and produced higher inflammatory cytokines (IL-8, IFN-γ and TNF-α) compared to those challenged with SA14. In mechanism, we found the integration of PHB21 genome caused the upregulated expression of many genes encoding products involved in bacterial biofilm formation, adherence to host cells, anti-phagocytosis, and virulence. This study may provide novel knowledge of “bacteria-phage-interactions” in MRSA.

The Bacillus phage SPβ has been known for about 50 years, but only a few strains are available. We isolated four new wild-type strains of the SPbeta species. Phage vB_BsuS-Goe14 introduces its prophage into the spoVK locus, previously not observed to be used by SPβ-like phages. Sequence data revealed the genome replication strategy and the genome packaging mode of SPβ-like phages. We extracted 55 SPβ-like prophages from public Bacillus genomes, thereby discovering three more integration loci and one additional type of integrase. The identified prophages resemble four new species clusters and three species orphans in the genus Spbetavirus. The determined core proteome of all SPβ-like prophages consists of 38 proteins. The integration cassette proved to be not conserved, even though, present in all strains. It consists of distinct integrases. Analysis of SPβ transcriptomes revealed three conserved genes, yopQ, yopR, and yokI, to be transcribed from a dormant prophage. While yopQ and yokI could be deleted from the prophage without activating the prophage, damaging of yopR led to a clear-plaque phenotype. Under the applied laboratory conditions, the yokI mutant showed an elevated virion release implying the YokI protein being a component of the arbitrium system.

The novel bacterial virus Mimir87, infecting the salt-tolerant bacterium Virgibacillus halotolerans, was isolated from worker honey bees. Mimir87 has an elongated head and a long non-contractile tail consistent with members of the Siphoviridae phage family. The phage genome comprises 48,016 base pairs and encodes 68 predicted proteins, to 34 of which a function could be assigned from homology analysis. The phage encodes two metabolism-related transporter proteins previously not observed in bacteriophage genomes. Mimir87 displays some relatedness to several Bacillus and Paenibacillus viruses; however, the overall sequence dissimilarity suggests Mimir87 to be a representative of a new phage genus.

Staphylococcus aureus is a biofilm-producing organism that is frequently isolated from various environments worldwide. Because of the natural resistance of S. aureus biofilm to antibiotics, bacteriophages are considered as a promising alternative for its removal. The bacteriophage vB_SauS_JS02 was isolated from livestock wastewater and showed activity against multidrug-resistant S. aureus. The phage vB_SauS_JS02 exhibited a broad host range and possessed a large burst size (52 PFU/CFU) as well as moderate pH stability (4–11) and appropriate thermal tolerance (40–50°C). Electron microscopy and genome sequence revealed that vB_SauS_JS02 belonged to Triavirus genus in Siphoviridae family. Genetic analysis of the 46 kb sequence of vB_SauS_JS02 revealed 66 ORFs. The predicted protein products of the ORFs were clustered functionally into five groups as follows: replication/regulation, DNA packaging, structure/morphogenesis, lysis and lysogeny. Although the phage vB_SauS_JS02 was a temperate phage, it exhibited a higher inhibiting and degrading activity against planktonic cells (80~90% reduction), even to S. aureus biofilm (~68% reduction in biofilm formation). Moreover, the removal activity of the phage vB_SauS_JS02 against both planktonic cells and S. aureus biofilms was even better than that of the antibiotic (ceftazidime). In summary, the present study introduced the phage vB_SauS_JS02 as a potential biocontrol agent against biofilm-producing S. aureus after making it virulent. It may be applicable for phage therapy.

In this study, the genomes of two lytic bacteriophages, vB_EcoS-phiEc3 and vB_EcoS-phiEc4, were sequenced and characterized using bioinformatics approaches. Whole-genome analysis showed that both phages belonged to the Kagunavirus genus, Guernseyvirinae subfamily and Siphoviridae family. Moreover, their genomes had 45, 288 bp and 44,540 bp, and G + C content of 48.42% and 50.04%, respectively. The genome of vB_EcoS-phiEc3 harbored 80 protein coding sequences (CDSs), whereas vB_EcoS-phiEc4 harbored 75 CDSs. Among them, 50 CDSs in vB_EcoS-phiEc3 and 44 CDSs in vB_EcoS-phiEc4 were considered as functional genes. Their lytic activity against multidrug-resistant uropathogenic Escherichia coli (UPEC) strains, as well as the absence of antibiotic resistance genes, lysogenic and virulence genes, enable vB_EcoS-phiEc3 and vB_EcoS-phiEc4 as a safe therapy option against UPEC infections.

Phytopathogenic pseudomonads are widespread in the world and cause a wide range of plant diseases. In this work, we describe the Pseudomonas phage Pf-10, which is a part of the biopesticide “Multiphage” used for bacterial diseases of agricultural crops caused by Pseudomonas syringae. The Pf-10 chromosome is a dsDNA molecule with two direct terminal repeats (DTRs). The phage genomic DNA is 39,424 bp long with a GC-content of 56.5%. The Pf-10 phage uses a packaging mechanism based on T7-like short DTRs, and the length of each terminal repeat is 257 bp. Electron microscopic analysis has shown that phage Pf-10 has the podovirus morphotype. Phage Pf-10 is highly stable at pH values from 5 to 10 and temperatures from 4 to 60 °C and has a lytic activity against Pseudomonas strains. Phage Pf-10 is characterized by fast adsorption rate (80% of virions attach to the host cells in 10 min), but has a relatively small number of progeny (37 ± 8.5 phage particles per infected cell). According to the phylogenetic analysis, phage Pf-10 can be classified as a new phage species belonging to the genus Pifdecavirus, subfamily Studiervirinae, family Autographiviridae, order Caudovirales.

As antibiotic resistance is being a threat to public health worldwide, bacteriophages are re-highlighted as alternative antimicrobials to fight with pathogens. Various wild-type phages isolated from diverse sources have been tested, but potential mutant phages generated by genome engineering or random mutagenesis are drawing increasing attention. Here, we applied a chelating agent, sodium pyrophosphate, to the staphylococcal temperate Siphoviridae phage SA3821 to introduce random mutations. Through 30 sequential sodium pyrophosphate challenges and random selections, the suspected mutant phage SA3821M was isolated. SA3821M maintained an intact virion morphology, but exhibited better bactericidal activity against its host Staphylococcous aureus CCARM 3821 for up to 17 h and thermostability than its parent, SA3821. Sodium pyrophosphate-mediated mutations in SA3821M were absent in lysogenic development genes but concentrated (83.9%) in genes related to the phage tail, particularly in the tail tape measure protein, indicating that changes in the tail module might have been responsible for the altered traits. This intentional random mutagenesis through controlled treatments with sodium pyrophosphate could be applied to other phages as a simple but potent method to improve their traits as alternative antimicrobials.

A novel Lactobacillus delbrueckii bacteriophage PMBT4 was isolated from the Nigerian fermented milk product nono. The phage possesses a long and thin, non-contractile tail and an isometric head, indicating that it belongs to the Siphoviridae family. A neck passage structure (`collar`), previously hypothesized to be encoded by two genes located in the Lactobacillus delbrueckii phage LL-K insertion sequence (KIS) element, as well as in two additional Lb. delbrueckii phages Ld17 and Ld25A, could also be observed on an estimated 1-5% of phage particles by transmission electron microscopy. However, neither mapping of high throughput sequencing data to KIS element genes from Lb. delbrueckii phages LL-K, Ld17 and Ld25A nor PCR amplification of the KIS element genes could corroborate the presence of these genes in the PMBT4 genome. The PMBT4 genome consists of 31,399 bp with a mol% GC content of 41.6 and exhibits high (95-96%) sequence homologies to Lb. delbrueckii phages c5, Ld3, Ld25A and Ld17, which assigned PMBT4 as a new member of this genus, i.e. the Cequinquevirus genus.

Background: Based on the WHO, multidrug-resistant Klebsiella pneumoniae is a priority pathogen that causes opportunistic infections and is widely spread in the environment. Phage therapy is considered a natural, safe, and very efficient alternative to treat difficult-to-treat infections. Objectives: This study aimed to isolate highly virulent, lytic bacteriophages and evaluate their efficacy for lysing multidrug-resistant K. pneumoniae. Methods: Municipal wastewater samples were collected and filtered using 0.22 µm syringe filters and cultivated with log-phase cultures of K. pneumoniae using enrichment media. After 48 h of incubation, the cultures were centrifuged, and the resultant supernatant was filtered (0.22 µm). The detection of the phage was done using the spot assay with K. pneumoniae as the host. One-step growth kinetics and bacterial reduction tests were conducted to assess the growth kinetics of the isolated phage. The stability of the isolated phage was characterized by subjecting it to various temperature and pH conditions. The chemical stability of the K. pneumoniae phage was determined by exposing it to various organic compounds. A panel of 20 bacterial strains was tested using the spot assay, as well as double agar overlying assay, to determine the host range of the isolated phage. Results: Out of 40 wastewater samples tested, only one sample was tested positive for the K. pneumoniae phage (2.5%) that was lytic against the host strain. The K. pneumoniae phage had a latent period of 15 min and a burst size of 100 virions per infected cell. It was most stable at 37°C and pH range of 6.0 to 10.0. Chemically, the K. pneumoniae phage was resistant to 10% chloroform treatment. Transmission electron micrograph indicated that the K. pneumoniae phage belonged to the order Caudovirales, family Siphoviridae, morphotype B1. Conclusions: Most of the characteristic features of the K. pneumoniae phage indicated the potential of this phage to be used in phage therapy. Hence, a comprehensive study is highly recommended to characterize the K. pneumoniae phage genome, detect its molecular interactions with the host cell, and determine its lytic activity in combination with other phages, which may lead to the efficient utilization of this phage in phage therapy against K. pneumoniae infections.

Bacteriophages play important roles in determining bacterial communities, including plant microbiota. Here, we describe four lytic phages, three Siphoviridae and one Podoviridae , isolated from four different bacterial species found on the leaves of horse chestnut trees. Their double-stranded DNA (dsDNA) genomes range from 39,095 to 46,062 bp and contain 51 to 70 genes.

Patients with Coronavirus Disease 2019 (COVID-19), due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection mainly present with respiratory issues and related symptoms, in addition to significantly affected digestive system, especially the intestinal tract. While several studies have shown changes in the intestinal flora of patients with COVID-19, not much information is available on the gut virome of such patients. In this study, we used the viromescan software on the latest gut virome database to analyze the intestinal DNA virome composition of 15 patients with COVID-19 and investigated the characteristic alternations, particularly of the intestinal DNA virome to further explore the influence of COVID-19 on the human gut. The DNA viruses in the gut of patients with COVID-19 were mainly crAss-like phages (35.48%),Myoviridae(20.91%), andSiphoviridae(20.43%) family of viruses. Compared with healthy controls, the gut virome composition of patients with COVID-19 changed significantly, especially the crAss-like phages family, from the first time of hospital admission. A potential correlation is also indicated between the change in virome and bacteriome (likeTectiviridaeandBacteroidaceae). The abundance of the viral and bacterial population was also analyzed through continuous sample collection from the gut of patients hospitalized due to COVID-19. The gut virome is indeed affected by the SARS-CoV-2 infection, and along with gut bacteriome, it may play an important role in the disease progression of COVID-19. These conclusions would be helpful in understanding the gut-related response and contribute to the treatment and prevention strategies of COVID-19.

We characterized the complete genome sequence of actinobacteriophage Yavru ( Siphoviridae ), a cluster FE bacteriophage infecting Arthrobacter globiformis NRRL B-2979; it was 89.5% identical to cluster FE phage Whytu, with a capsid width of 50 nm and a tail length of 90 nm. The genome was 15,193 bp in length, with 23 predicted protein-coding genes.

Yersinia enterocolitica is a food-borne Gram-negative pathogen responsible for several gastrointestinal disorders. Host-specific lytic bacteriophages have been increasingly used recently as an alternative or complementary treatment to combat bacterial infections, especially when antibiotics fail. Here, we describe the proteogenomic characterization and host receptor identification of the siphovirus vB_YenS_phi R2-01 (in short, phi R2-01) that infects strains of several Yersinia enterocolitica serotypes. The phi R2-01 genome contains 154 predicted genes, 117 of which encode products that are homologous to those of Escherichia bacteriophage T5. The phi R2-01 and T5 genomes are largely syntenic, with the major differences residing in areas encoding hypothetical phi R2-01 proteins. Label-free mass-spectrometry-based proteomics confirmed the expression of 90 of the phi R2-01 genes, with 88 of these being either phage particle structural or phage-particle-associated proteins. In vitro transposon-based host mutagenesis and phi R2-01 adsorption experiments identified the outer membrane vitamin B12 receptor BtuB as the host receptor. This study provides a proteogenomic characterization of a T5-type bacteriophage and identifies specific Y. enterocolitica strains sensitive to infection with possible future applications of phi R2-01 as a food biocontrol or phage therapy agent.

Mycobacteriophages are phages that infect mycobacteria resulting in their killing. Although lysis is the primary mechanism by which mycobacteriophages cause cell death, others such as abortive infection may also be involved. We took recourse to perform immunofluorescence and electron microscopic studies using mycobacteriophage D29 infected Mycobacterium smegmatis cells to investigate this issue. We could observe the intricate details of the infection process using these techniques such as adsorption, the phage tail penetrating the thick mycolic acid layer, formation of membrane pores, membrane blebbing, and phage release. We observed a significant increase in DNA fragmentation and membrane depolarization using cell-biological techniques symptomatic of programmed cell death (PCD). As Toxin-Antitoxin (TA) systems mediate bacterial PCD, we measured their expression profiles with and without phage infection. Of the three TAs examined, MazEF, VapBC, and phd/doc, we found that in the case of VapBC, a significant decrease in the antitoxin (VapB): toxin (VapC) ratio was observed following phage infection, implying that high VapC may have a role to play in the induction of mycobacterial apoptotic cell death following phage infection. This study indicates that D29 infection causes mycobacteria to undergo morphological and molecular changes that are hallmarks of apoptotic cell death.

The mycobacteriophages JeTaime (E cluster) and Luna22 (Q cluster) were isolated from soil in Providence, Rhode Island, and Charleston, South Carolina, respectively, using a Mycobacterium smegmatis mc 2 155 host. The genome of JeTaime is 75,099 bp (142 predicted genes), and that of Luna22 is 53,730 bp (87 predicted genes). Both phages exhibit Siphoviridae morphology.

Extra-intestinal Escherichia coli express several virulence factors that increase their ability to colonize and survive in different localizations. The K1 capsular type is involved in several infections, including meningitis, urinary tract, and bloodstream infections. The aims of this work were to isolate, characterize, and assess the in vivo efficacy of phages targeting avian pathogenic E. coli (APEC) O18:K1, which shares many similarities with the human strains responsible for neonatal meningitis. Eleven phages were isolated against APEC O18:K1, and four of them presenting a narrow spectrum targeting E. coli K1 strains were further studied. The newly isolated phages vB_EcoS_K1-ULINTec2 were similar to the Siphoviridae family, and vB_EcoP_K1-ULINTec4, vB_EcoP_K1-ULINTec6, and vB_EcoP_K1-ULINTec7 to the Autographiviridae family. They are capsular type (K1) dependent and present several advantages characteristic of lytic phages, such as a short adsorption time and latent period. vB_EcoP_K1-ULINTec7 is able to target both K1 and K5 strains. This study shows that these phages replicate efficiently, both in vitro and in vivo in the Galleria mellonella model. Phage treatment increases the larvae survival rates, even though none of the phages were able to eliminate the bacterial load.

Phage-based biocontrol of bacteria is considered a natural approach to combat foodborne pathogens. Salmonella spp. are notifiable and highly prevalent pathogens that cause foodborne diseases worldwide. In this study, six bacteriophages were isolated and further characterized that infect food-derived Salmonella isolates from different meat sources. The siphovirus VB_StyS-LmqsSP1, which was isolated from a cow’s nasal swab, was further subjected to in-depth characterization. Phage-host interaction investigations in liquid medium showed that vB_StyS-LmqsSP1 can suppress the growth of Salmonella species isolates at 37°C for 10 h and significantly reduce the bacterial titer at 4°C. A reduction of 1.4 to 3 log units was observed in investigations with two food-derived Salmonella isolates and one reference strain under cooling conditions using multiplicities of infection (MOIs) of 10(4) and 10(5). Phage application on chicken skin resulted in a reduction of about 2 log units in the tested Salmonella isolates from the first 3 h throughout a 1-week experiment at cooling temperature and with an MOI of 10(5). The one-step growth curve analysis using vB_StyS-LmqsSP1 demonstrated a 60-min latent period and a burst size of 50 to 61 PFU/infected cell for all tested hosts. Furthermore, the genome of the phage was determined to be free from genes causing undesired effects. Based on the phenotypic and genotypic properties, LmqsSP1 was assigned as a promising candidate for biocontrol of Salmonella enterica serovar Typhimurium in food. IMPORTANCE Salmonella enterica is one of the major global causes of foodborne enteritis in humans. The use of chemical sanitizers for reducing bacterial pathogens in the food chain can result in the spread of bacterial resistance. Targeted and clean-label intervention strategies can reduce Salmonella contamination in food. The significance of our research demonstrates the suitability of a bacteriophage (vB_StyS-LmqsSP1) for biocontrol of Salmonella enterica serovar Typhimurium on poultry due to its lytic efficacy under conditions prevalent in food production environments.

Escherichia phage 590B, which was isolated from community sewage water in Chandigarh, India, exhibited lytic activity against an extensively drug-resistant uropathogenic Escherichia coli isolate. The genome of the phage is linear, double-stranded, and 44.39 kb long. Phage 590B is a member of the Siphoviridae family and is closest to phage vB_EcoS_XY2, which was isolated in China.

We present here the results of the analysis of the complete genome sequence of a potentially temperate phage, vB_Sb_QDWS, which was isolated from wastewater samples collected in Qingdao, China. The genome of phage vB_Sb_QDWS is composed of a double-stranded DNA that is 47,902 bp in length with a G + C content of 63.16%. It is predicted to contain 69 putative protein-encoding genes. Microscopic and genomic analysis showed that vB_Sb_QDWS is a novel phage of the class Siphoviridae.

Klebsiella pneumoniae is an important pathogen that can infect both humans and cattle. The widespread K. pneumoniae and its high drug resistance make it difficult to treat Klebsiella infections/diseases. In this study, a lytic K. pneumoniae bacteriophage vB_Kpn_B01 was isolated from a dairy farm trough in Sichuan Province, and its biological properties were studied, and the entire genome of vB_Kpn_B01 was sequenced. The therapeutic effects of the phage on disease-causing mice were preliminarily tested. Phages found in this study are double-stranded DNA bacterial viruses belonging to the family Siphoviridae, Sugarlandvirus. The results suggest that vB_Kpn_B01 has strong specificity and low adaptability to different adverse conditions. Meanwhile, the predicted gene products of phage vB_Kpn_B01 comprised 149 coding sequences (CDS) and 25 tRNAs, of which 34 CDS had known functions. Of course, vB_Kpn_B01 did not contain any known antibiotic-resistant or virulent genes. The pathological sections of the liver and lungs of mice showed that the inflammatory scores of the treatment group were lower than in the bacterial group. Phage vB_Kpn_B01 alleviated the inflammatory response in the organs of the infected mice, and the organ tissue bacterial load of the treatment group was significantly lower than that of the bacterial group. Therefore, vB_Kpn_B01 can inhibit the proliferation of K. pneumoniae 18 in vivo and can alleviate the inflammation of target organs caused by infectious bacteria, which preliminarily indicates that vB_Kpn_B01 has a certain therapeutic effect on laboratory-infected mice.

18 pages, 5 figures, 2 tables, supplementary material https://www.frontiersin.org/articles/10.3389/fmicb.2021.701414/full#supplementary-material.-- Data Availability Statement: All DNA sequence FASTQ files are deposited in the NCBI Nucleotide Archive under SRR14023866 (CB2 Bacterial and Archaeal), SRR 14023937 (GSL 3510 Bacterial and Archaeal), SRR14023877 (GB14 Bacterial and Archaeal), SRR14023878 (CB2 Viral), SRR14023865 (GSL3510 Viral), and SRR14023938 (GB14 Viral). The Bioproject id is PRJNA714934. The NCBI genome accession numbers for the MAGs and viral assemblies are shown in Supplementary Table S10, Viruses play vital biogeochemical and ecological roles by (a) expressing auxiliary metabolic genes during infection, (b) enhancing the lateral transfer of host genes, and (c) inducing host mortality. Even in harsh and extreme environments, viruses are major players in carbon and nutrient recycling from organic matter. However, there is much that we do not yet understand about viruses and the processes mediated by them in the extreme environments such as hypersaline habitats. The Great Salt Lake (GSL) in Utah, United States is a hypersaline ecosystem where the biogeochemical role of viruses is poorly understood. This study elucidates the diversity of viruses and describes virus–host interactions in GSL sediments along a salinity gradient. The GSL sediment virosphere consisted of Haloviruses (32.07 ± 19.33%) and members of families Siphoviridae (39.12 ± 19.8%), Myoviridae (13.7 ± 6.6%), and Podoviridae (5.43 ± 0.64%). Our results demonstrate that salinity alongside the concentration of organic carbon and inorganic nutrients (nitrogen and phosphorus) governs the viral, bacteria, and archaeal diversity in this habitat. Computational host predictions for the GSL viruses revealed a wide host range with a dominance of viruses that infect Proteobacteria, Actinobacteria, and Firmicutes. Identification of auxiliary metabolic genes for photosynthesis (psbA), carbon fixation (rbcL, cbbL), formaldehyde assimilation (SHMT), and nitric oxide reduction (NorQ) shed light on the roles played by GSL viruses in biogeochemical cycles of global relevance, The funding for this project was provided by the United States National Science Foundation with project number 1510255, With the institutional support of the ‘Severo OchoaCentre of Excellence’ accreditation (CEX2019-000928-S)

Background Marine bacteriophages play key roles in the community structure of microorganisms, biogeochemical cycles, and the mediation of genetic diversity through horizontal gene transfer. Recently, traditional isolation methods, complemented by high-throughput sequencing metagenomics technology, have greatly increased our understanding of the diversity of bacteriophages. Oceanospirillum, within the order Oceanospirillales, are important symbiotic marine bacteria associated with hydrocarbon degradation and algal blooms, especially in polar regions. However, until now there has been no isolate of an Oceanospirillum bacteriophage, and so details of their metagenome has remained unknown. Results Here, we reported the first Oceanospirillum phage, vB_OliS_GJ44, which was assembled into a 33,786 bp linear dsDNA genome, which includes abundant tail-related and recombinant proteins. The recombinant module was highly adapted to the host, according to the tetranucleotides correlations. Genomic and morphological analyses identified vB_OliS_GJ44 as a siphovirus, however, due to the distant evolutionary relationship with any other known siphovirus, it is proposed that this virus could be classified as the type phage of a new Oceanospirivirus genus within the Siphoviridae family. vB_OliS_GJ44 showed synteny with six uncultured phages, which supports its representation in uncultured environmental viral contigs from metagenomics. Homologs of several vB_OliS_GJ44 genes have mostly been found in marine metagenomes, suggesting the prevalence of this phage genus in the oceans. Conclusions These results describe the first Oceanospirillum phage, vB_OliS_GJ44, that represents a novel viral cluster and exhibits interesting genetic features related to phage–host interactions and evolution. Thus, we propose a new viral genus Oceanospirivirus within the Siphoviridae family to reconcile this cluster, with vB_OliS_GJ44 as a representative member.

Therapeutic bacteriophages, commonly called as phages, are a promising potential alternative to antibiotics in the management of bacterial infections of a wide range of organisms including cultured fish. Their natural immunogenicity often induces the modulation of a variated collection of immune responses within several types of immunocytes while promoting specific mechanisms of bacterial clearance. However, to achieve standardized treatments at the practical level and avoid possible side effects in cultivated fish, several improvements in the understanding of their biology and the associated genomes are required. Interestingly, a particular feature with therapeutic potential among all phages is the production of lytic enzymes. The use of such enzymes against human and livestock pathogens has already provided in vitro and in vivo promissory results. So far, the best-understood phages utilized to fight against either Gram-negative or Gram-positive bacterial species in fish culture are mainly restricted to the Myoviridae and Podoviridae, and the Siphoviridae, respectively. However, the current functional use of phages against bacterial pathogens of cultured fish is still in its infancy. Based on the available data, in this review, we summarize the current knowledge about phage, identify gaps, and provide insights into the possible bacterial control strategies they might represent for managing aquaculture-related bacterial diseases.

Acinetobacter baumannii is a nosocomial pathogen, which is a problem worldwide due to the emergence of a difficult-to-treat multidrug-resistant A. baumannii (MDRAB). Endolysins are hydrolytic enzymes produced by a bacteriophage that can be used as a potential therapeutic agent for multidrug-resistant bacterial infection in replacing antibiotics. Here, we isolated a novel bacteriophage through prophage induction using mitomycin C from clinical A. baumannii 1656-2. Morphologically, ΦAb1656-2 was identified as a Siphoviridae family bacteriophage, which can infect MDRAB. The whole genome of ΦAb1656-2 was sequenced, and it showed that it is 50.9 kb with a G + C content of 38.6% and 68 putative open reading frames (ORFs). A novel endolysin named AbEndolysin with an N-acetylmuramidase-containing catalytic domain was identified, expressed, and purified from ΦAb1656-2. Recombinant AbEndolysin showed significant antibacterial activity against MDRAB clinical strains without any outer membrane permeabilizer. These results suggest that AbEndolysin could represent a potential antimicrobial agent for treating MDRAB clinical isolates.

Multidrug-resistant bacteria have become a major threat to global public health. Bacteriophages are regarded as a promising substitute. Here, we present a novel lytic Stenotrophomonas maltophilia phage, vB_SM_ytsc_ply2008005c, which was isolated from sewage water samples in Qingdao, east China. Virion morphology of phage particles indicated that ply2008005c has an icosahedral head (56 ± 5 nm in diameter) and a noncontractile sheathed tail (129 ± 6 nm in length), which are the typical characteristics of phages belonging to the family Siphoviridae. Phage ply2008005c could be used for phage therapy for its stability in a wide pH (4 to 12) range and high temperature (up to 70°C) environment. Genome analysis revealed that ply2008005c has a circular double-strand DNA of 42,318 bp with a G+C content of 63.02%. It shared the closest relationship with phage vB_PaeS_PAO1_Ab18, but the homology coverage is just 20%. There were 54 open reading frames predicted in its genome, including three unique proteins and 34 functional genes in different modules. The phylogenetic analysis revealed that ply2008005c forms a distinct branch of the family Siphoviridae. These results demonstrated that ply2008005c was supposed to be a representative new member within the family Siphoviridae, which could be considered a potential bioagent against multidrug-resistant S. maltophilia.

The emerging carbapenem resistance and extended spectrum β-lactamases (ESBL) producing strains of Klebsiella pneumoniae, are one of the critical pathogens for which novel therapeutic alternatives are required on urgent basis. Biofilm formation further aids in virulence due to impermeable nature. Bacteriophages are bacterial predators which due to their selective, and nontoxic nature can be used as an alternate to counter MDR infections. Hence, the current study was intended to isolate, characterize, and develop phage cocktail as a possible therapy against ESBL K. pneumoniae.The two-novel bacteriophage A¥L and A¥M were isolated from environmental samples and characterized for host specificity and physicochemical stability (i.e., temperature and pH). Isolated phages alone or together as cocktail was further evaluated for in vitro biofilm eradication and infection clearance using in vivo murine model. Whole genome sequencing was performed for identification, evolutionary relationship, and bioinformatics analysis.The isolated phage A¥L and A¥M belonged to Myoviridae and Siphoviridae family, respectively and showed good thermal (-20, 37, 45, and 60̊C) and pH (5, 7, 9, and 11) stability. At MOI of 0.001, both phages displayed short eclipse period of 5 and 10 min, respectively. Phages alone or together as cocktail showed 50-70% eradication of 48 h mature biofilm. Majority of the cells within biofilm was found dead as evinced from live dead staining. Atomic force and scanning electron microscopic analysis showed distorted biofilm with ruptured cells. The isolated phage or their cocktail significantly inhibited K. pneumoniae associated mortality in intraperitoneal inoculated mice model CONCLUSION: These findings imply that the phage might be a good option for eliminating K. pneumoniae infections and further studies could help in development of these phage as a bio-control product.