Isolation and characterization of a phage collection against Pseudomonas putida.

The environmental bacterium, Pseudomonas putida, possesses a broad spectrum of metabolic pathways. This makes it highly promising for use in biotechnological production as a cell factory, as well as in bioremediation strategies to degrade various aromatic pollutants. For P. putida to flourish in its environment, it must withstand the continuous threats posed by bacteriophages. Interestingly, until now, only a handful of phages have been isolated for the commonly used laboratory strain, P. putida KT2440, and no phage defence mechanisms have been characterized. In this study, we present a new Collection of Environmental P. putida Phages from Estonia, or CEPEST. This collection comprises 67 double-stranded DNA phages, which belong to 22 phage species and 9 phage genera. Our findings reveal that most phages in the CEPEST collection are more infectious at lower temperatures, have a narrow host range, and require an intact lipopolysaccharide for P. putida infection. Furthermore, we show that cryptic prophages present in the P. putida chromosome provide strong protection against the infection of many phages. However, the chromosomal toxin-antitoxin systems do not play a role in the phage defence of P. putida. This research provides valuable insights into the interactions between P. putida and bacteriophages, which could have significant implications for biotechnological and environmental applications.

Evaluating the efficacy of a phage cocktail against Pseudomonas fluorescens group strains in raw milk: microbiological, physical, and chemical analyses.

The objective of this study was to investigate the effectiveness of a phage cocktail against Pseudomonas fluorescens group and its effect on the microbial, physical and chemical properties of raw milk during different storage conditions. A phage cocktail consisting of Pseudomonas fluorescens, Pseudomonas tolaasii, and Pseudomonas libanensis phages was prepared. As a result, reductions in fluorescent Pseudomonas counts of up to 3.44 log units for the storage at 4 °C and 2.38 log units for the storage at 25 °C were achieved. Following the phage application, it is found that there was no significant difference in the total mesophilic aerobic bacteria and Enterobacteriaceae counts. However, it was observed that the number of lactic acid bacteria was higher in phage-treated groups. The results also showed that pH values in the phage added groups were lower than the others and the highest titratable acidity was obtained only in the bacteria-inoculated group. As a future perspective, this study suggests that, while keeping the number of target microorganisms under control in the milk with the use of phages during storage, the microbiota and accordingly the quality parameters of the milk can be affected. This work contributes to the development of effective strategies for maintaining the quality and extending the shelf life of milk and dairy products.

Characteristics and whole-genome analysis of a novel Pseudomonas syringae pv. tomato bacteriophage D6 isolated from a karst cave.

Pseudomonas syringae is a gram-negative plant pathogen that infects plants such as tomato and poses a threat to global crop production. In this study, a novel lytic phage infecting P. syringae pv. tomato DC3000, named phage D6, was isolated and characterized from sediments in a karst cave. The latent period of phage D6 was found to be 60 min, with a burst size of 16 plaque-forming units per cell. Phage D6 was stable at temperatures between 4 and 40 °C but lost infectivity when heated to 70 °C. Its infectivity was unaffected at pH 6-10 but became inactivated at pH ≤ 5 or ≥ 12. The genome of phage D6 is a linear double-stranded DNA of 307,402 bp with a G + C content of 48.43%. There is a codon preference between phage D6 and its host, and the translation of phage D6 gene may not be entirely dependent on the tRNA library provided by the host. A total of 410 open reading frames (ORFs) and 14 tRNAs were predicted in its genome, with 92 ORFs encoding proteins with predicted functions. Phage D6 showed low genomic similarity to known phage genomes in the GenBank and Viral sequence databases. Genomic and phylogenetic analyses revealed that phage D6 is a novel phage. The tomato plants were first injected with phage D6, and subsequently with Pst DC3000, using the foliar spraying and root drenching inoculum approach. Results obtained after 14 days indicated that phage D6 inoculation decreased P. syringae-induced symptoms in tomato leaves and inhibited the pathogen's growth in the leaves. The amount of Pst DC3000 was reduced by 150- and 263-fold, respectively. In conclusion, the lytic phage D6 identified in this study belongs to a novel phage within the Caudoviricetes class and has potential for use in biological control of plant diseases.

Sewage and sewage-contaminated environments are the most prominent sources to isolate phages against Pseudomonas aeruginosa.

BACKGROUND: P. aeruginosa is the primary source of hospital-acquired infections. Unfortunately, antibiotic resistance is growing to precariously high levels, making the infections by this pathogen life-threatening and hard to cure. One possible alternative to antibiotics is to use phages. However, the isolation of phages suitable for phage therapy- be lytic, be efficient, and have a broad host range -against some target bacteria has proven difficult. To identify the best places to look for these phages against P. aeruginosa we screened hospital sewages, soils, and rivers in two cities. RESULTS: We isolated eighteen different phages, determined their host range, infection property, and plaque morphology. We found that the sewage and sewage-contaminated environments are the most reliable sources for the isolation of Pseudomonas phages. In addition, phages isolated from hospital sewage showed the highest efficiency in lysing the bacteria used for host range determination. In contrast, phages from the river had larger plaque size and lysed bacteria with higher levels of antibiotic resistance. CONCLUSIONS: Our findings provided additional support for the importance of sewage as the source of phage isolation.

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.

Complete genome sequence of Phobos: a novel bacteriophage with unusual genomic features that infects Pseudomonas syringae.

The GenBank database contains over 2580 complete genome sequences from bacteriophages. However, limited reports are available concerning phages can that lyse members of Pseudomonas syringae, although this is a widespread bacterial species that can infect almost 200 plant species. In the present study, we isolated and characterized a new Siphoviridae phage, named "Pseudomonas phage vB_PsyS_Phobos" (for brevity, referred to here as Phobos). To our knowledge, this is one of the first genome sequences reported for a phage with lytic activity against P. syringae pv. syringae. The genome of Phobos is dsDNA of 56,734 bp with a GC content of 63.3%, containing 65 ORFs. Genome analysis revealed that Phobos is a novel lytic phage with unique genomic features and low similarity to other phages, suggesting that Phobos represents a new phage genus. Genome sequencing did not reveal sequences with significant similarity to known virulence factors, antibiotic resistance genes, potential immunoreactive allergens, or lysogeny-related proteins, suggesting suggests that phage Phobos is strictly lytic. Therefore, Phobos may be suitable for formulation as a biocontrol agent against P. syringae pv. syringae.

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

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

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

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

Characterization of a bacteriophage with broad host range against strains of Pseudomonas aeruginosa isolated from domestic animals.

BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen and one of the leading causes of nosocomial infections. Moreover, the species can cause severe infections in cystic fibrosis patients, in burnt victims and cause disease in domestic animals. The control of these infections is often difficult due to its vast repertoire of mechanisms for antibiotic resistance. Phage therapy investigation with P. aeruginosa bacteriophages has aimed mainly the control of human diseases. In the present work, we have isolated and characterized a new bacteriophage, named Pseudomonas phage BrSP1, and investigated its host range against 36 P. aeruginosa strains isolated from diseased animals and against P. aeruginosa ATCC strain 27853. RESULTS: We have isolated a Pseudomonas aeruginosa phage from sewage. We named this virus Pseudomonas phage BrSP1. Our electron microscopy analysis showed that phage BrSP1 had a long tail structure found in members of the order Caudovirales. "In vitro" biological assays demonstrated that phage BrSP1 was capable of maintaining the P. aeruginosa population at low levels for up to 12 h post-infection. However, bacterial growth resumed afterward and reached levels similar to non-treated samples at 24 h post-infection. Host range analysis showed that 51.4% of the bacterial strains investigated were susceptible to phage BrSP1 and efficiency of plating (EOP) investigation indicated that EOP values in the strains tested varied from 0.02 to 1.72. Analysis of the phage genome revealed that it was a double-stranded DNA virus with 66,189 bp, highly similar to the genomes of members of the genus Pbunavirus, a group of viruses also known as PB1-like viruses. CONCLUSION: The results of our "in vitro" bioassays and of our host range analysis suggested that Pseudomonas phage BrSP1 could be included in a phage cocktail to treat veterinary infections. Our EOP investigation confirmed that EOP values differ considerably among different bacterial strains. Comparisons of complete genome sequences indicated that phage BrSP1 is a novel species of the genus Pbunavirus. The complete genome of phage BrSP1 provides additional data that may help the broader understanding of pbunaviruses genome evolution.

Isolation and characterization of a bacteriophage and its potential to disrupt multi-drug resistant Pseudomonas aeruginosa biofilms.

A lytic Pseudomonas aeruginosa bacteriophage, vB_PaeM_LS1, was isolated and characterized herein. To examine the eligibility of bacteriophage vB_PaeM_LS1 as a therapeutic bacteriophage, we analysed its genome and compared it to similar bacteriophages. Genome of bacteriophage vB_PaeM_LS1 consisted of a linear, double-stranded DNA molecule 66,095 bp in length and with 55.7% G + C content. Neighbor-joining analysis of the large subunit terminase showed that bacteriophage vB_PaeM_LS1 had similarity to the Pbunavirus genus. The potential of the lytic bacteriophage to disrupt Pseudomonas aeruginosa biofilms was assessed by scanning electron microscopy and bacterial counts. This study revealed that the bacteriophage vB_PaeM_LS1 with its lytic effect showed a high potential impact on the inhibition of the growth of Pseudomonas aeruginosa biofilm formation.

Complete genome sequences of Aeromonas and Pseudomonas phages as a supportive tool for development of antibacterial treatment in aquaculture.

BACKGROUND: Aquaculture is the fastest growing sector of food production worldwide. However, one of the major reasons limiting its effectiveness are infectious diseases among aquatic organisms resulting in vast economic losses. Fighting such infections with chemotherapy is normally used as a rapid and effective treatment. The rise of antibiotic resistance, however, is limiting the efficacy of antibiotics and creates environmental and human safety concerns due to their massive application in the aquatic environment. Bacteriophages are an alternative solution that could be considered in order to protect fish against pathogens while minimizing the side-effects for the environment and humans. Bacteriophages kill bacteria via different mechanisms than antibiotics, and so fit nicely into the 'novel mode of action' concept desired for all new antibacterial agents. METHODS: The bacteriophages were isolated from sewage water and characterized by RFLP, spectrum of specificity, transmission electron microscopy (TEM) and sequencing (WGS). Bioinformatics analysis of genomic data enables an in-depth characterization of phages and the choice of phages. This allows an optimised choice of phage for therapy, excluding those with toxin genes, virulence factor genes, and genes responsible for lysogeny. RESULTS: In this study, we isolated eleven new bacteriophages: seven infecting Aeromonas and four infecting Pseudomonas, which significantly increases the genomic information of Aeromonas and Pseudomonas phages. Bioinformatics analysis of genomic data, assessing the likelihood of these phages to enter the lysogenic cycle with experimental data on their specificity towards large number of bacterial field isolates representing different locations. CONCLUSIONS: From 11 newly isolated bacteriophages only 6 (25AhydR2PP, 50AhydR13PP, 60AhydR15PP, 22PfluR64PP, 67PfluR64PP, 71PfluR64PP) have a potential to be used in phage therapy due to confirmed lytic lifestyle and absence of virulence or resistance genes.

Complete genome sequence of PFP1, a novel T7-like Pseudomonas fluorescens bacteriophage.

The complete genomic sequence of Pseudomonas fluorescens bacteriophage PFP1, isolated from sewage samples collected in Liaoning Province, China, were sequenced in this study and found to be 40,914 bp long. The PFP1 genome is composed of linear double-stranded DNA with 55.81% G+C content and 45 putative protein-coding genes, and no rRNA and tRNA genes. Comparative genomics and phylogenetic analysis revealed that the Pseudomonas fluorescens phage PFP1 is a new member of the genus T7virus. This information can be used to develop novel phage-based control strategies against Pseudomonas fluorescens.

Genomic characterization and phylogenetic analysis of the novel Pseudomonas phage PPSC2.

We isolated a Pseudomonas phage infecting Pseudomonas fluorescens SA1 separated from a soil sample collected in Sichuan Province, China. This phage, which we named PPSC2, has a genome that is composed of a 97,330-bp-long linear double-stranded DNA with 47.51% G+C content and 168 putative protein-coding genes. We identified 20 tRNA genes in the genome of PPSC2, and the tRNA GC content ranged from 44.2% to 58.4%. Phylogenetic and BLASTn analysis revealed that the Pseudomonas phage PPSC2 should be considered a new member of the family Myoviridae.

Three novel Pseudomonas phages isolated from composting provide insights into the evolution and diversity of tailed phages.

BACKGROUND: Among viruses, bacteriophages are a group of special interest due to their capacity of infecting bacteria that are important for biotechnology and human health. Composting is a microbial-driven process in which complex organic matter is converted into humus-like substances. In thermophilic composting, the degradation activity is carried out primarily by bacteria and little is known about the presence and role of bacteriophages in this process. RESULTS: Using Pseudomonas aeruginosa as host, we isolated three new phages from a composting operation at the Sao Paulo Zoo Park (Brazil). One of the isolated phages is similar to Pseudomonas phage Ab18 and belongs to the Siphoviridae YuA-like viral genus. The other two isolated phages are similar to each other and present genomes sharing low similarity with phage genomes in public databases; we therefore hypothesize that they belong to a new genus in the Podoviridae family. Detailed genomic descriptions and comparisons of the three phages are presented, as well as two new clusters of phage genomes in the Viral Orthologous Clusters database of large DNA viruses. We found sequences encoding homing endonucleases that disrupt a putative ribonucleotide reductase gene and an RNA polymerase subunit 2 gene in two of the phages. These findings provide insights about the evolution of two-subunits RNA polymerases and the possible role of homing endonucleases in this process. Infection tests on 30 different strains of bacteria reveal a narrow host range for the three phages, restricted to P. aeruginosa PA14 and three other P. aeruginosa clinical isolates. Biofilm dissolution assays suggest that these phages could be promising antimicrobial agents against P. aeruginosa PA14 infections. Analyses on composting metagenomic and metatranscriptomic data indicate association between abundance variations in both phage and host populations in the environment. CONCLUSION: The results about the newly discovered and described phages contribute to the understanding of tailed bacteriophage diversity, evolution, and role in the complex composting environment.

A carrier state is established in Pseudomonas aeruginosa by phage LeviOr01, a newly isolated ssRNA levivirus.

ssRNA bacteriophages are very abundant but poorly studied, particularly in relation to their effect on bacterial evolution. We isolated a new Pseudomonas aeruginosa levivirus, vB_PaeL_PcyII-10_LeviOr01, from hospital waste water. Its genome comprises 3669 nucleotides and encodes four putative proteins. Following bacterial infection, a carrier state is established in a fraction of the cells, conferring superinfection immunity. Such cells also resist other phages that use type IV pili as a receptor. The carrier population is composed of a mixture of cells producing phage, and susceptible cells that are non-carriers. Carrier cells accumulate phage until they burst, releasing large quantities of virions. The continuous presence of phage favours the emergence of host variants bearing mutations in genes involved in type IV pilus biogenesis, but also in genes affecting lipopolysaccharide (LPS) synthesis. The establishment of a carrier state in which phage particles are continuously released was previously reported for some dsRNA phages, but has not previously been described for a levivirus. The present results highlight the importance of the carrier state, an association that benefits both phages and bacteria and plays a role in bacterial evolution.

Preparation of Tc99m-Labeled Pseudomonas Bacteriophage without Adversely Impacting Infectivity or Biodistribution.

Bacteriophages (phages) are ubiquitous viruses which have adapted to infect and replicate within target bacteria, their only known hosts, in a strain specific fashion with minimal cross infectivity. The recent steep rise in antibiotic resistance throughout the world has renewed interest in adapting phages for the imaging and treatment of bacterial infection in humans. In this article, we describe the current limitations surrounding the radiolabeling of phage for the imaging and treatment of bacterial infection and methods to overcome these difficulties. Specifically, we examined the effects of hydrazinonicotinamide conjugation and removal of bacterial DNA on the infectivity, biodistribution, and radionuclide imaging of a phage lytic for a clinically relevant strain of Pseudomonas aeruginosa, a common Gram-negative bacterial pathogen often resistant to multiple antibiotics. We found that all but the briefest reaction of concentrated phage with hydrazinonicotinamide (≤3 min) resulted in nearly complete loss of infectivity. Furthermore, we determined that digestion and removal of bacterial DNA was needed to avoid high nonspecific uptake of hydrazinonicotinamide-labeled phage within the liver and spleen as well as prolonged circulation in the blood. We also demonstrate the surprisingly wide soft tissue and organ biodistribution and rapid pharmacokinetics of 99mTc-hydrazinonicotinamide-labeled phage in normal mice as well as its imaging characteristics and efficacy in wounded mice infected with bioluminescent Pseudomonas aeruginosa. In conclusion, the preservation of phage infectivity and removal of all bacterial containments including DNA are critical methodologic considerations in the labeling of phages for imaging and therapy.

Complete genome sequences of two novel autographiviruses infecting a bacterium from the Pseudomonas fluorescens group.

In this paper, we describe two independent isolates of a new member of the subfamily Autographivirinae, Pseudomonas phage KNP. The type strain (KNP) has a linear, 40,491-bp-long genome with GC content of 57.3%, and 50 coding DNA sequences (CDSs). The genome of the second strain (WRT) contains one CDS less, encodes a significantly different tail fiber protein and is shorter (40,214 bp; GC content, 57.4%). Phylogenetic analysis indicates that both KNP and WRT belong to the genus T7virus. Together with genetically similar Pseudomonas phages (gh-1, phiPSA2, phiPsa17, PPPL-1, shl2, phi15, PPpW-4, UNO-SLW4, phiIBB-PF7A, Pf-10, and Phi-S1), they form a divergent yet coherent group that stands apart from the T7-like viruses (sensu lato). Analysis of the diversity of this group and its relatedness to other members of the subfamily Autographivirinae led us to the conclusion that this group might be considered as a candidate for a new genus.

Isolation and characterization of wetland VSW-3, a novel lytic cold-active bacteriophage of Pseudomonas fluorescens.

Wetlands are often called the "kidneys of the Earth" and contribute substantially to environmental improvement. Pseudomonas fluorescens is a major contaminant of milk products and causes the spoilage of refrigerated foods and fresh poultry. In this study, we isolated and characterized a lytic cold-active bacteriophage named VSW-3 together with P. fluorescens SW-3 cells from the Napahai wetland in China. Electron microscopy showed that VSW-3 had an icosahedral head (56 nm) and a tapering tail (20 nm × 12 nm) and a genome size of approximate 40 kb. On the basis of the top-scoring hits in the BLASTP analysis, VSW-3 showed a high degree of module similarity to the Pseudomonas phages Andromeda and Bf7. The latent and burst periods were 45 and 20 min, respectively, with an average burst size of 90 phage particles per infected cell. The pH and thermal stability of VSW-3 were also explored. The optimal pH was found to be 7.0 and the activity decreased rapidly when the temperature exceeded 60 °C. VSW-3 is a cold-active bacteriophage, hence, it is important to research its ability to prevent product contamination caused by P. fluorescens and to characterize its relationship with its host P. fluorescens in the future.

Isolation of Polyvalent Bacteriophages by Sequential Multiple-Host Approaches.

Many studies on phage biology are based on isolation methods that may inadvertently select for narrow-host-range phages. Consequently, broad-host-range phages, whose ecological significance is largely unexplored, are consistently overlooked. To enhance research on such polyvalent phages, we developed two sequential multihost isolation methods and tested both culture-dependent and culture-independent phage libraries for broad infectivity. Lytic phages isolated from activated sludge were capable of interspecies or even interorder infectivity without a significant reduction in the efficiency of plating (0.45 to 1.15). Two polyvalent phages (PX1 of the Podoviridae family and PEf1 of the Siphoviridae family) were characterized in terms of adsorption rate (3.54 × 10(-10) to 8.53 × 10(-10) ml/min), latent time (40 to 55 min), and burst size (45 to 99 PFU/cell), using different hosts. These phages were enriched with a nonpathogenic host (Pseudomonas putida F1 or Escherichia coli K-12) and subsequently used to infect model problematic bacteria. By using a multiplicity of infection of 10 in bacterial challenge tests, >60% lethality was observed for Pseudomonas aeruginosa relative to uninfected controls. The corresponding lethality for Pseudomonas syringae was ∼ 50%. Overall, this work suggests that polyvalent phages may be readily isolated from the environment by using different sequential hosts, and this approach should facilitate the study of their ecological significance as well as enable novel applications.

Characterization of Pseudomonas aeruginosa phage KPP21 belonging to family Podoviridae genus N4-like viruses isolated in Japan.

Bacteriophages (phages) belonging to the family Podoviridae genus N4-like viruses have been used as therapeutic agent in phage therapy against Pseudomonas aeruginosa infections. P. aeruginosa phage KPP21 was isolated in Japan, and phylogenetically investigated the phages belonging to this viral genus. Morphological and genetic analyses confirmed that phage KPP21 belongs to the family Podoviridae genus N4-like viruses. Moreover, phylogenetic analyses based on putative DNA polymerase and major virion protein showed that P. aeruginosa phages belonging to the genus N4-like viruses are separated into two lineages and that phage KPP21 is in the same clade as phage LUZ7.