Isolation and characterization of two Staphylococcus aureus lytic bacteriophages "Huma" and "Simurgh".

Nowadays finding the new antimicrobials is necessary due to the emerging of multidrug resistant strains. The present study aimed to isolate and characterize bacteriophages against S. aureus. Strains Huma and Simurgh were the two podovirus morphology phages which isolated and then characterized. Huma and Simurgh had a genome size of 16,853 and 17,245 bp, respectively and both were Rosenblumvirus with G + C content of 29%. No lysogeny-related genes, nor virulence genes were identified in their genomes. They were lytic only against two out of four S. aureus strains. They also were able to inhibit S. aureus for 8 h in-vitro. Both showed a rapid adsorption. Huma and Simurgh had the latent period of 80 and 60 m and the burst sizes of 45 and 40 PFU/ml and also, they showed very low cell toxicity of 1.23%-1.79% on HT-29 cells, respectively. Thus, they can be considered potential candidates for biocontrol applications.

Characterization of the Novel Phage vB_VpaP_FE11 and Its Potential Role in Controlling Vibrio parahaemolyticus Biofilms.

Vibrio parahaemolyticus causes aquatic vibriosis. Its biofilm protects it from antibiotics; therefore, a new different method is needed to control V. parahaemolyticus for food safety. Phage therapy represents an alternative strategy to control biofilms. In this study, the lytic Vibrio phage vB_VpaP_FE11 (FE11) was isolated from the sewers of Guangzhou Huangsha Aquatic Market. Electron microscopy analysis revealed that FE11 has a typical podovirus morphology. Its optimal stability temperature and pH range were found to be 20-50 °C and 5-10 °C, respectively. It was completely inactivated following ultraviolet irradiation for 20 min. Its latent period is 10 min and burst size is 37 plaque forming units/cell. Its double-stranded DNA genome is 43,397 bp long, with a G + C content of 49.24% and 50 predicted protein-coding genes. As a lytic phage, FE11 not only prevented the formation of biofilms but also could destroy the formed biofilms effectively. Overall, phage vB_VpaP_FE11 is a potential biological control agent against V. parahaemolyticus and the biofilm it produces.

Isolation of the Novel Phage PHB09 and Its Potential Use against the Plant Pathogen Pseudomonas syringae pv. actinidiae.

Bacteriophages are viruses that specifically infect target bacteria. Recently, bacteriophages have been considered potential biological control agents for bacterial pathogens due to their host specificity. Pseudomonas syringae pv. actinidiae (Psa) is a reemerging pathogen that causes bacterial canker of kiwifruit (Actinidia sp.). The economic impact of this pest and the development of resistance to antibiotics and copper sprays in Psa and other pathovars have led to investigation of alternative management strategies. Phage therapy may be a useful alternative to conventional treatments for controlling Psa infections. Although the efficacy of bacteriophage φ6 was evaluated for the control of Psa, the characteristics of other DNA bacteriophages infecting Psa remain unclear. In this study, the PHB09 lytic bacteriophage specific to Psa was isolated from kiwifruit orchard soil. Extensive host range testing using Psa isolated from kiwifruit orchards and other Pseudomonas strains showed PHB09 has a narrow host range. It remained stable over a wide range of temperatures (4-50 °C) and pH values (pH 3-11) and maintained stability for 50 min under ultraviolet irradiation. Complete genome sequence analysis indicated PHB09 might belong to a new myovirus genus in Caudoviricetes. Its genome contains a total of 94,844 bp and 186 predicted genes associated with phage structure, packaging, host lysis, DNA manipulation, transcription, and additional functions. The isolation and identification of PHB09 enrich the research on Pseudomonas phages and provide a promising biocontrol agent against kiwifruit bacterial canker.

A highly specific Serratia-infecting T7-like phage inhibits biofilm formation in two different genera of the Enterobacteriaceae family.

Due to the emergence of multidrug-resistant bacteria, bacteriophages have become a viable alternative in controlling bacterial growth or biofilm formation. Biofilm is formed by extracellular polymeric substances (EPS) and is one of the factors responsible for increasing bacterial resistance. Bacteriophages have been studied as a bacterial control agent by use of phage enzymes or due to their bactericidal activities. A specific phage against Serratia marcescens was isolated in this work and was evaluated its biological and genomic aspects. The object of this study was UFV01, a bacteriophage belonging to the Podoviridae family, genus Teseptimavirus (group of lytic viruses), specific to the species S. marcescens, which may be related to several amino acid substitutions in the virus tail fibers. Despite this high specificity, the phage reduced the biofilm formation of several Escherichia coli strains without infecting them. UFV01 presents a relationship with phages of the genus Teseptimavirus, although it does not infect any of the E. coli strains evaluated, as these others do. All the characteristics make the phage an interesting alternative in biofilm control in hospital environments since small breaks in the biofilm matrix can lead to a complete collapse.

Revealing the Viral Community in the Hadal Sediment of the New Britain Trench.

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

Isolation and characterization of the novel Pseudomonas stutzeri bacteriophage 8P.

Bacteriophage 8P was isolated with a Pseudomonas stutzeri strain isolated from an oil reservoir as its host bacterium. The phage genome comprises 63,753 base pairs with a G+C content of 64.35. The phage encodes 63 predicted proteins, and 27 of them were functionally assigned. No tRNA genes were found. Comparative genomics analysis showed that 8P displayed some relatedness to F116-like phages (78% identity, 20% query coverage). The genome has very low sequence similarity to the other phage genomes in the GenBank database and Viral Sequence Database. Based on whole-genome analysis and transmission electron microscopy imaging, 8P is proposed to be a member of a new species in the genus Hollowayvirus, family Podoviridae.

Extraordinary diversity of viruses in deep-sea sediments as revealed by metagenomics without prior virion separation.

Our current knowledge of the virosphere in deep-sea sediments remains rudimentary. Here we investigated viral diversity at both gene and genomic levels in deep-sea sediments of Southwest Indian Ocean. Analysis of 19 676 106 non-redundant genes from the metagenomic DNA sequences revealed a large number of unclassified viral groups in these samples. A total of 1106 high-confidence viral contigs were obtained after two runs of assemblies, and 217 of these contigs with sizes up to ~120 kb were shown to represent complete viral genomes. These contigs are clustered with no known viral genomes, and over 2/3 of the ORFs on the viral contigs encode no known functions. Furthermore, most of the complete viral contigs show limited similarity to known viral genomes in genome organization. Most of the classified viral contigs are derived from dsDNA viruses belonging to the order Caudovirales, including primarily members of the families Myoviridae, Podoviridae and Siphoviridae. Most of these viruses infect Proteobacteria and, less frequently, Planctomycetes, Firmicutes, Chloroflexi, etc. Auxiliary metabolic genes (AMGs), present in abundance on the viral contigs, appear to function in modulating the host ability to sense environmental gradients and community changes, and to uptake and metabolize nutrients.

Biological characteristics and genome analysis of a novel phage vB_KpnP_IME279 infecting Klebsiella pneumoniae.

Klebsiella pneumoniae (family Enterobacteriaceae) is a gram-negative bacterium that has strong pathogenicity to humans and can cause sepsis, pneumonia, and urinary tract infection. In recent years, the unreasonable use of antibacterial drugs has led to an increase in drug-resistant strains of K. pneumoniae, a serious threat to public health. Bacteriophages, viruses that infect bacteria, are ubiquitous in the natural environment. They are considered to be the most promising substitute for antibiotics because of their high specificity, high efficiency, high safety, low cost, and short development cycle. In this study, a novel phage designated vB_KpnP_IME279 was successfully isolated from hospital sewage using a multidrug-resistant strain of K. pneumoniae as an indicator. A one-step growth curve showed that vB_KpnP_IME279 has a burst size of 140 plaque-forming units/cell and a latent period of 20 min at its optimal multiplicity of infection (MOI = 0.1). Phage vB_KpnP_IME279 survives in a wide pH range between 3 and 11 and is stable at temperatures ranging from 40 to 60 °C. Ten of the 20 strains of K. pneumoniae including the host bacteria were lysed by the phage vB_KpnP_IME279, and the multilocus sequence typing and wzi typing of the 10 strains were ST11, ST37, ST375, wzi209, wzi52, and wzi72, respectively. The genome of vB_KpnP_IME279 is 42,518 bp long with a G + C content of 59.3%. Electron microscopic observation showed that the phage belongs to the family Podoviridae. BLASTN alignment showed that the genome of the phage has low similarity with currently known phages. The evolutionary relationship between phage vB_KpnP_IME279 and other Podoviridae was analyzed using a phylogenetic tree based on sequences of phage major capsid protein and indicates that the phage vB_KpnP_IME279 belongs to the Podoviridae subfamily. These data enhance understanding of K. pneumoniae phages and will help in development of treatments for multidrug-resistant bacteria using phages.

Novel Caulobacter bacteriophages illustrate the diversity of the podovirus genus Rauchvirus.

The podovirus BPP-1 is currently the only member of the Podovirus genus Rauchvirus. Here, we describe three new Caulobacter bacteriophages (Jess A, SR18, and RW) that show genetic similarity to BPP-1 but have many different genetic and structural features that differentiate them from BPP-1. Jess A and SR18 are closely related to each other and should be considered two members of a new species. They share a similar gene order with BPP-1. However, they do not appear to form lysogens or have the tropism switching mechanism that has been described for BPP-1. Bacteriophage RW also exhibits some homology to BPP-1. However, it is quite different from the other three phages, and we propose that it should be considered a representative of a third species of the genus Rauchvirus. Taken together, the differences among these four members of the genus Rauchvirus indicate that this divergent genus has a long evolutionary history and that there are many more rauchviruses waiting to be discovered.

Isolation, characterization, and efficacy of bacteriophages isolated against Citrobacter spp. an in vivo approach in a zebrafish model (Danio rerio).

Citrobacter infections are becoming an increasingly significant health problem in aquaculture in South-Eastern countries. The objective of this study was to isolate and evaluate the potential of lytic bacteriophages against Citrobacter infections. TEM analysis revealed that the isolated phages Citrophage MRM19 and Citrophage MRM57 were identified to be Siphovirus and Podovirus family of the order Caudovirales. The phage life-cycle studies showed that Citrophage MRM19 had an adsorption time of 18 ± 1 min and a latency period of 25 ± 3 min with burst size of 110 ± 20 phages/infected cell and Citrophage MRM57 had an adsorption time of 15 ± 1 min and a latency period of 25 ± 2 min with burst size of 50 ± 5 phages/infected cell. In vitro studies indicated that the bacterial load was reduced by 5 and 7 log units within 12 h by Citrophage MRM19 and Citrophage MRM57. The in vivo efficacy of the phages was studied using zebrafish (Danio rerio) as a model organism in low-scale tanks. The study unveiled that the use of phages increased the survival up to 17%, 23%, and 26% in the case of Citrophage MRM19, Citrophage MRM57, and phage cocktail treatment, respectively. Our study indicated that bacteriophages are suitable biocontrol agents against Citrobacter spp. especially in aquaculture industry.

Isolation and characterization of bacteriophages against virulent Aeromonas hydrophila.

BACKGROUND: Aeromonas hydrophila is an important water-borne pathogen that leads to a great economic loss in aquaculture. Along with the abuse of antibiotics, drug-resistant strains rise rapidly. In addition, the biofilms formed by this bacterium limited the antibacterial effect of antibiotics. Bacteriophages have been attracting increasing attention as a potential alternative to antibiotics against bacterial infections. RESULTS: Five phages against pathogenic A. hydrophila, named N21, W3, G65, Y71 and Y81, were isolated. Morphological analysis by transmission electron microscopy revealed that phages N21, W3 and G65 belong to the family Myoviridae, while Y71 and Y81 belong to the Podoviridae. These phages were found to have broad host spectra, short latent periods and normal burst sizes. They were sensitive to high temperature but had a wide adaptability to the pH. In addition, the phages G65 and Y81 showed considerable bacterial killing effect and potential in preventing formation of A. hydrophila biofilm; and the phages G65, W3 and N21 were able to scavenge mature biofilm effectively. Phage treatments applied to the pathogenic A. hydrophila in mice model resulted in a significantly decreased bacterial loads in tissues. CONCLUSIONS: Five A. hydrophila phages were isolated with broad host ranges, low latent periods, and wide pH and thermal tolerance. And the phages exhibited varying abilities in controlling A. hydrophila infection. This work presents promising data supporting the future use of phage therapy.

A Tailspike with Exopolysaccharide Depolymerase Activity from a New Providencia stuartii Phage Makes Multidrug-Resistant Bacteria Susceptible to Serum-Mediated Killing.

Providencia stuartii is emerging as a significant drug-resistant nosocomial pathogen, which encourages the search for alternative therapies. Here, we have isolated Providencia stuartii phage Stuart, a novel podovirus infecting multidrug-resistant hospital isolates of this bacterium. Phage Stuart is a proposed member of a new Autographivirinae subfamily genus, with a 41,218-bp genome, direct 345-bp repeats at virion DNA ends, and limited sequence similarity of proteins to proteins in databases. Twelve out of the 52 predicted Stuart proteins are virion components. We found one to be a tailspike with depolymerase activity. The tailspike could form a highly thermostable oligomeric ß-structure migrating close to the expected trimer in a nondenaturing gel. It appeared to be essential for the infection of three out of four P. stuartii hosts infected by phage Stuart. Moreover, it degraded the exopolysaccharide of relevant phage Stuart hosts, making the bacteria susceptible to serum killing. Prolonged exposure of a sensitive host to the tailspike did not cause the emergence of bacteria resistant to the phage or to serum killing, opposite to the prolonged exposure to the phage. This indicates that phage tail-associated depolymerases are attractive antivirulence agents that could complement the immune system in the fight with P. stuartiiIMPORTANCE The pace at which multidrug-resistant strains emerge has been alarming. P. stuartii is an infrequent but relevant drug-resistant nosocomial pathogen causing local to systemic life-threatening infections. We propose an alternative approach to fight this bacterium based on the properties of phage tailspikes with depolymerase activity that degrade the surface bacterial polymers, making the bacteria susceptible to the immune system. Unlike antibiotics, phage tailspikes have narrow and specific substrate spectra, and by acting as antivirulent but not bactericidal agents they do not cause the selection of resistant bacteria.

Discovery of Three Toxic Proteins of Klebsiella Phage fHe-Kpn01.

The lytic phage, fHe-Kpn01 was isolated from sewage water using an extended-spectrum beta-lactamase-producing strain of Klebsiella pneumoniae as a host. The genome is 43,329 bp in size and contains direct terminal repeats of 222 bp. The genome contains 56 predicted genes, of which proteomics analysis detected 29 different proteins in purified phage particles. Comparison of fHe-Kpn01 to other phages, both morphologically and genetically, indicated that the phage belongs to the family Podoviridae and genus Drulisvirus. Because fHe-Kpn01 is strictly lytic and does not carry any known resistance or virulence genes, it is suitable for phage therapy. It has, however, a narrow host range since it infected only three of the 72 tested K. pneumoniae strains, two of which were of capsule type KL62. After annotation of the predicted genes based on the similarity to genes of known function and proteomics results on the virion-associated proteins, 22 gene products remained annotated as hypothetical proteins of unknown function (HPUF). These fHe-Kpn01 HPUFs were screened for their toxicity in Escherichia coli. Three of the HPUFs, encoded by the genes g10, g22, and g38, were confirmed to be toxic.

Unravelling the consequences of the bacteriophages in human samples.

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

Genomic characterization of a novel virulent phage infecting Shigella fiexneri and isolated from sewage.

Shigella fiexneri phage SGF2 is a novel lytic phage isolated from a sewage sample. Morphological characterization indicates that phage SGF2 is a member of the Podoviridae family, producing virions with an isometric head (82.6 ± 8 nm diameter) and a short non-contractile tail (length 52 ± 8 nm). This phage specifically infected the Shigella fiexneri. One-step growth curves indicated that the burst period of phage SGF2 is 30 min, with an approximate burst size of 38. The full-length genome was sequenced and potential virulence genes were detected. We will discuss the potential application of phage SGF2 in phage therapy.

Complete genome sequence of Xanthomonas phage RiverRider, a novel N4-like bacteriophage that infects the strawberry pathogen Xanthomonas fragariae.

Xanthomonas phage RiverRider is a novel N4-like bacteriophage and the first phage isolated from the plant pathogen Xanthomonas fragariae. Electron microscopy revealed a Podoviridae morphology consisting of isometric heads and short noncontractile tails. The complete genome of RiverRider is 76,355 bp in length, with 90 open reading frames and seven tRNAs. The genome is characteristic of N4-like bacteriophages in both content and organization, having predicted proteins characterized into the functional groups of transcription, DNA metabolism, DNA replication, lysis, lysis inhibition, structure and DNA packaging. Amino acid sequence comparisons for proteins in these categories showed highest similarities to well-characterized N4-like bacteriophages isolated from Achromobacter xylosoxidans and Erwinia amylovora. However, the tail fiber proteins of RiverRider are clearly distinct from those of other N4-like phages. RiverRider was able to infect seven different strains of X. fragariae and none of the other species of Xanthomonas tested.

Isolation and application of bacteriophages alone or in combination with nisin against planktonic and biofilm cells of Staphylococcus aureus.

Staphylococcus aureus is a notorious foodborne pathogen since it has ability to produce variety of toxins including heat-stable enterotoxin, form biofilm, and acquire resistance to antibiotics. Biocontrol of foodborne pathogens by lytic bacteriophages garners increasing interest from both researchers and food industry. In the present study, 29 phages against S. aureus were successfully isolated from chicken, pork, and fish. Characterization of the isolates revealed that phage SA46-CTH2 belonging to Podoviridae family had a number of features suitable for food industry applications such as wide host range, short latent period, large burst size, high stress tolerance, and a genome free of virulence genes. Furthermore, phage SA46-CTH2 alone or in combination with nisin exhibited great efficacy in reducing planktonic and biofilm cells of S. aureus at various conditions tested. The combination of phage SA46-CTH2 and nisin was also found to be able to inhibit the regrowth of S. aureus at both 37 and 24 °C.Key points• A total of 29 S. aureus phages were successfully isolated from fish, pork, and chicken products. • Phage SA46-CTH2 was characterized by host range, morphology, and genome sequencing. • SA46-CTH2 significantly reduced both planktonic and biofilm cells of S. aureus. • Combination of SA46-CTH2 and nisin inhibited the regrowth of S. aureus.

Characterization of Salmonella bacteriophages and their potential use in dishwashing materials.

AIMS: The aims of this study were to isolate and characterize novel Salmonella phages and to evaluate the effectiveness of phage cocktails used as antibacterial agents in dishwashing materials. METHODS AND RESULTS: The effective phages, vB_STy-RN5i1 and vB_STy-RN29, were isolated from drain water samples collected from open markets using Salmonella Typhimurium as the host strain. These phages were identified as members of Podoviridae and Siphoviridae, respectively. Both phages infected at least six Salmonella serovars and rapidly lysed their host within one hour. They were stable at 4-45°C and at pH 6-9 for at least an hour while being evaluated in this study. The phage application results indicated that bacterial cells were reduced by 3⋅1 and 2⋅7 log CFU per ml at room temperature when they encountered the phage cocktail on scouring pads (SPs) and dishwashing sponges (DSs), respectively. CONCLUSIONS: The isolated Salmonella phages, vB_STy-RN5i1 and vB_STy-RN29, had potential against Salm. Typhimurium and could reduce the occurrence of bacterial-cross-contamination from dishwashing materials, which have been reported to be a source of bacteria, to other kitchen utensils and food. SIGNIFICANCE AND IMPACT OF THE STUDY: The successful reduction of bacterial contamination in dishwashing materials by the phage cocktail consisting of vB_STy-RN5i1 and vB_STy-RN29 reveals its potential to be an alternative antimicrobial agent for SPs and DSs.

Isolation and Characterization of AbTJ, an Acinetobacter baumannii Phage, and Functional Identification of Its Receptor-Binding Modules.

A. baumannii is an opportunistic pathogen and a major cause of various community-acquired infections. Strains of this species can be resistant to multiple antimicrobial agents, leaving limited therapeutic options, also lacking in methods for accurate and prompt diagnosis. In this context, AbTJ, a novel phage that infects A. baumannii MDR-TJ, was isolated and characterized, together with its two tail fiber proteins. Morphological analysis revealed that it belongs to Podoviridae family. Its host range, growth characteristics, stability under various conditions, and genomic sequence, were systematically investigated. Bioinformatic analysis showed that AbTJ consists of a circular, double-stranded 42670-bp DNA molecule which contains 62 putative open reading frames (ORFs). Genome comparison revealed that the phage AbTJ is related to the Acinetobacter phage Ab105-1phi (No. KT588074). Tail fiber protein (TFPs) gp52 and gp53 were then identified and confirmed as species-specific proteins. By using a combination of bioluminescent methods and magnetic beads, these TFPs exhibit excellent specificity to detect A. baumannii. The findings of this study can be used to help control opportunistic infections and to provide pathogen-binding modules for further construction of engineered bacteria of diagnosis and treatment.

Isolation and Characterization of a Novel Bacteriophage Infecting Carbapenem-Resistant Klebsiella pneumoniae.

A novel virulent phage, vB_KpnP_IME337, isolated from a hospital sewage in Beijing, China, that infects carbapenem-resistant Klebsiella pneumoniae KN2 capsular type was identified and characterized. Next-generation sequencing and genome analysis revealed that vB_KpnP_IME337 had a linear double-stranded genome with a length of 44,266 base pairs and G+C content of 53.7%. Fifty-two putative open reading frames were identified, and no transfer RNA-encoding genes were detected. BLASTn analysis revealed that phage vB_KpnP_IME337 had the highest sequence similarity with Klebsiella phage phiBO1E, with genome coverage of 79%. Based on morphology, phage vB_KpnP_IME337 was determined to belong to the family Podoviridae of the order Caudovirales. It was shown that phage vB_KpnP_IME337 had an infection duration of ~ 90 min and 10 min latent period, and a highly specific to host strain. In conclusion, phage vB_KpnP_IME337 may be a promising alternative candidate to antibiotic treatment for controlling diseases caused by drug-resistant K. pneumoniae.