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.

Preliminary survey of local bacteriophages with lytic activity against multi-drug resistant bacteria.

Bacteriophages (phages) represent a potential alternative for combating multi-drug resistant bacteria. Because of their narrow host range and the ever emergence of novel pathogen variants the continued search for phages is a prerequisite for optimal treatment of bacterial infections. Here we performed an ad hoc survey in the surroundings of a University hospital for the presence of phages with therapeutic potential. To this end, 16 aquatic samples of different origins and locations were tested simultaneously for the presence of phages with lytic activity against five current, but distinct strains each from the ESKAPE-group (i.e., Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae). Phages could be isolated for 70% of strains, covering all bacterial species except S. aureus. Apart from samples from two lakes, freshwater samples were largely devoid of phages. By contrast, one liter of hospital effluent collected at a single time point already contained phages active against two-thirds of tested strains. In conclusion, phages with lytic activity against nosocomial pathogens are unevenly distributed across environments with the prime source being the immediate hospital vicinity.

Expanding the Phage Galaxy: Isolation and Characterization of Five Novel Streptomyces Siphoviruses Ankus, Byblos, DekoNeimoidia, Mandalore, and Naboo.

Background: Key features of the actinobacterial genus Streptomyces are multicellular, filamentous growth, and production of a broad portfolio of bioactive molecules. These characteristics appear to play an important role in phage-host interactions and are modulated by phages during infection. To accelerate research of such interactions and the investigation of novel immune systems in multicellular bacteria, phage isolation, sequencing, and characterization are needed. This is a prerequisite for establishing systematic collections that appropriately cover phage diversity for comparative analyses. Material & Methods: As part of a public outreach program within the priority program SPP 2330, involving local schools, we describe the isolation and characterization of five novel Streptomyces siphoviruses infecting S. griseus, S. venezuelae, and S. olivaceus. Results: All isolates are virulent members of two existing genera and, additionally, establish a new genus in the Stanwilliamsviridae family. In addition to an extensive set of tRNAs and proteins involved in phage replication, about 80% of phage genes encode hypothetical proteins, underlining the yet underexplored phage diversity and genomic dark matter still found in bacteriophages infecting actinobacteria. Conclusions: Taken together, phages Ankus, Byblos, DekoNeimoidia, Mandalore, and Naboo expand the phage diversity and contribute to ongoing research in the field of Streptomyces phage-host interactions.

Genomic Characterization of Phage ZP3 and Its Endolysin LysZP with Antimicrobial Potential against Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) is a significant bacterial pathogen responsible for outbreaks of bacterial leaf blight in rice, posing a major threat to rice cultivation worldwide. Effective management of this pathogen is crucial for ensuring rice yield and food security. In this study, we identified and characterized a novel Xoo phage, ZP3, isolated from diseased rice leaves in Zhejiang, China, which may offer new insights into biocontrol strategies against Xoo and contribute to the development of innovative approaches to combat bacterial leaf blight. Transmission electron microscopy indicated that ZP3 had a short, non-contractile tail. Genome sequencing and bioinformatic analysis showed that ZP3 had a double-stranded DNA genome with a length of 44,713 bp, a G + C content of 52.2%, and 59 predicted genes, which was similar to other OP1-type Xoo phages belonging to the genus Xipdecavirus. ZP3's endolysin LysZP was further studied for its bacteriolytic action, and the N-terminal transmembrane domain of LysZP is suggested to be a signal-arrest-release sequence that mediates the translocation of LysZP to the periplasm. Our study contributes to the understanding of phage-Xoo interactions and suggests that phage ZP3 and its endolysin LysZP could be developed into biocontrol agents against this phytopathogen.

Isolation and characterization of a novel lytic Parabacteroides distasonis bacteriophage φPDS1 from the human gut.

The human gut microbiome plays a significant role in health and disease. The viral component (virome) is predominantly composed of bacteriophages (phages) and has received significantly less attention in comparison to the bacteriome. This knowledge gap is largely due to challenges associated with the isolation and characterization of novel gut phages, and bioinformatic hurdles such as the lack of a universal phage marker gene and the absence of sufficient numbers of homologs in viral databases. Here, we describe the isolation from human feces of a novel lytic phage with siphovirus morphology, φPDS1, infecting Parabacteroides distasonis APCS2/PD, and classified within a newly proposed Sagittacolavirus genus. In silico and biological characterization of this phage is presented in this study. Key to the isolation of φPDS1 was the antibiotic-driven selective enrichment of the bacterial host in a fecal fermenter. Despite producing plaques and lacking genes associated with lysogeny, φPDS1 demonstrates the ability to coexist in liquid culture for multiple days without affecting the abundance of its host. Multiple studies have shown that changes in Parabacteroides distasonis abundance can be linked to various disease states, rendering this novel phage-host pair and their interactions of particular interest.

Use of the Naturally Occurring Bacteriophage Grouping Model for the Design of Potent Therapeutic Cocktails.

The specificity of phages and their ability to evolve and overcome bacterial resistance make them potentially useful as adjuncts in the treatment of antibiotic-resistant bacterial infections. The goal of this study was to mimic a natural grouping of phages of interest and to evaluate the nature of their proliferation dynamics with bacteria. We have, for the first time, transferred naturally occurring phage groups directly from their sources of isolation to in vitro and identified 13 P. aeruginosa and 11 K. pneumoniae phages of 18 different genera, whose host range was grouped as 1.2-17%, 28-48% and 60-87%, using a large collection of P. aeruginosa (n = 102) and K. pneumoniae (n = 155) strains carrying different virulence factors and phage binding receptors. We introduced the interpretation model curve for phage liquid culturing, which allows easy and quick analysis of bacterial and phage co-proliferation and growth of phage-resistant mutants (PRM) based on qualitative and partially quantitative evaluations. We assayed phage lytic activities both individually and in 14 different cocktails on planktonic bacterial cultures, including three resistotypes of P. aeruginosa (PAO1, PA14 and PA7) and seven K. pneumoniae strains of different capsular serotypes. Based on the results, the natural phage cocktails designed and tested in this study largely performed well and inhibited PRM growth either synergistically or in proto-cooperation. This study contributes to the knowledge of phage behavior in cocktails and the formulation of therapeutic phage preparations. The paper also provides a detailed description of the methods of working with phages.

Characterization of a Diverse Collection of Salmonella Phages Isolated from Tennessee Wastewater.

Background: Salmonella enterica is one of the most prevalent bacterial foodborne pathogens. Salmonella phages are currently used in biocontrol applications and have potential for use as therapeutics. Materials and Methods: Phages were enriched and purified from a diversity of Salmonella host isolates. Morphology was determined with transmission electron microscopy, host ranges were characterized using an efficiency of plaquing assay, and comparative genomic analysis was performed to determine taxonomy. Results: Ten phages were isolated and characterized. Phages showed activity against 23 out of the 24 Salmonella serovars evaluated. Two phages also showed activity against Escherichia coli strain B. Phages belonged to five different genera (Ithacavirus, Gelderlandvirus, Kuttervirus, Tlsvirus, and Epseptimavirus), two established species, and eight novel species. Conclusions: The phages described here further demonstrate the diversity of S. enterica phages present in wastewater effluent. This work contributes a collection of characterized phages from eastern Tennessee that may be of use in future phage-based applications targeting S. enterica.

More's the Same-Multiple Hosts Do Not Select for Broader Host Range Phages.

Bacteriophage host range is a result of the interactions between phages and their hosts. For phage therapy, phages with a broader host range are desired so that a phage can infect and kill the broadest range of pathogen strains or related species possible. A common, but not well-tested, belief is that using multiple hosts during the phage isolation will make the isolation of broader host range phage more likely. Using a Bacillus cereus group system, we compared the host ranges of phages isolated on one or four hosts and found that there was no difference in the breadth of host ranges of the isolated phages. Both narrow and broader host range phage were also equally likely to be isolated from either isolation procedure. While there are methods that reliably isolate broader host range phages, such as sequential host isolation, and there are other reasons to use multiple hosts during isolation, multiple hosts are not a consistent way to obtain broader host range phages.

Isolation, screening and characterization of phage.

Bacterial resistance threatens public health due to a lack of novel antibacterial classes since the 21st century. Bacteriophages, the most ubiquitous microorganism on Earth and natural predators of bacteria, have the potential to save the world from the post-antibiotic era. Therefore, phage isolation and characterization are in high demand to find suitable phages for therapeutic and bacterial control applications. The chapter presents brief guidance supported by recommendations on the isolation of phages, and initial screening of phage antimicrobial efficacy, in addition to, conducting comprehensive characterization addressing morphological, biological, genomic, and taxonomic features.

The Lytic Activity of Bacteriophage ZCSE9 against Salmonella enterica and Its Synergistic Effects with Kanamycin.

Salmonella, the causative agent of several diseases in humans and animals, including salmonellosis, septicemia, typhoid fever, and fowl typhoid, poses a serious threat to global public health and food safety. Globally, reports of therapeutic failures are increasing because of the increase in bacterial antibiotic resistance. Thus, this work highlights the combined phage-antibiotic therapy as a promising approach to combating bacterial resistance. In this manner, the phage ZCSE9 was isolated, and the morphology, host infectivity, killing curve, combination with kanamycin, and genome analysis of this phage were all examined. Morphologically, phage ZCSE9 is a siphovirus with a relatively broad host range. In addition, the phage can tolerate high temperatures until 80 °C with one log reduction and a basic environment (pH 11) without a significant decline. Furthermore, the phage prevents bacterial growth in the planktonic state, according to the results of the time-killing curve. Moreover, using the phage at MOI 0.1 with kanamycin against five different Salmonella serotypes reduces the required antibiotics to inhibit the growth of the bacteria. Comparative genomics and phylogenetic analysis suggested that phage ZCSE9, along with its close relatives Salmonella phages vB_SenS_AG11 and wksl3, belongs to the genus Jerseyvirus. In conclusion, phage ZCSE9 and kanamycin form a robust heterologous antibacterial combination that enhances the effectiveness of a phage-only approach for combating Salmonella.

Isolation of Novel Xanthomonas Phages Infecting the Plant Pathogens X. translucens and X. campestris.

The genus of Xanthomonas contains many well-known plant pathogens with the ability to infect some of the most important crop plants, thereby causing significant economic damage. Unfortunately, classical pest-control strategies are neither particularly efficient nor sustainable and we are, therefore, in demand of alternatives. Here, we present the isolation and characterization of seven novel phages infecting the plant-pathogenic species Xanthomonas translucens and Xanthomonas campestris. Transmission electron microscopy revealed that all phages show a siphovirion morphology. The analysis of genome sequences and plaque morphologies are in agreement with a lytic lifestyle of the phages making them suitable candidates for biocontrol. Moreover, three of the isolated phages form the new genus "Shirevirus". All seven phages belong to four distinct clusters underpinning their phylogenetic diversity. Altogether, this study presents the first characterized isolates for the plant pathogen X. translucens and expands the number of available phages for plant biocontrol.

In Vitro Techniques and Measurements of Phage Characteristics That Are Important for Phage Therapy Success.

Validated methods for phage selection, host range expansion, and lytic activity determination are indispensable for maximizing phage therapy outcomes. In this review, we describe some relevant methods, highlighting their advantages and disadvantages, and categorize them as preliminary or confirmatory methods where appropriate. Experimental conditions, such as the composition and consistency of culture media, have an impact on bacterial growth and, consequently, phage propagation and the selection of phage-resistant mutants. The phages require different experimental conditions to be tested to fully reveal their characteristics and phage therapy potential in view of their future use in therapy. Phage lytic activity or virulence should be considered as a result of the phage, its host, and intracellular/environmental factors, including the ability of a phage to recognize receptors on the bacterial cell surface. In vitro quantitative and qualitative measurements of phage characteristics, further validated by in vivo experiments, could be incorporated into one system or mathematical model/formula, which could predict a potential successful outcome of clinical applications.

Improving high-throughput techniques for bacteriophage discovery in multi-well plates.

Bacteriophages (also called phages) are viruses of bacteria that have numerous applications in medicine, agriculture, ecology, and molecular biology. With the increasing interest in phages for their many uses, it is now especially important to make phage discovery more efficient and economical. Using the host Mycobacterium smegmatis mc2155, which is a model organism for phage discovery research and is closely related to important pathogens of humans and other animals, we investigated three procedures that are an integral part of phage discovery: enrichment of environmental samples, phage isolation and detection (which can also be used for host range determination), and phage purification. Enrichment in 6-well plates was successful with most environmental samples, and enrichment in 24- and 96-well plates was successful with some environmental samples, demonstrating that larger sample volumes are preferred when possible, but smaller sample volumes may be acceptable if the starting concentration of phages is sufficiently high. Measuring absorbance in multi-well plates was at least as sensitive as the traditional plaque assay for the detection of phages. We also demonstrated a technique for the purification of single phage types from mixed cultures in liquid medium. Multi-well techniques can be used as alternatives or complementary approaches to traditional methods of phage discovery and characterization depending on the needs of the researcher in terms of time, available resources, host species, phage-bacteria matches, and specific goals. In the future, these techniques could be applied to the discovery of phages of aquatic mycobacteria and other hosts for which few phages have currently been isolated.

Bacteriophages: from Isolation to Application

Bacteriophages are considered as a potential alternative to fight pathogenic bacteria during the antibiotic resistance era. With their high specificity, they are widely used in various applications: medicine, food industry, agriculture, animal farms, biotechnology, diagnosis, etc. Many techniques have been designed by different researchers for phage isolation, purification, and amplification, each of which has strengths and weaknesses. However, all aim at having a reasonably pure phage sample that can be further characterized. Phages can be characterized based on their physiological, morphological or inactivation tests. Microscopy, in particular, opened a wide gate, not only for visualizing phage morphological structure, but also for monitoring biochemistry and behavior. Meanwhile, computational analysis of phage genomes provides more details about phage history, lifestyle, and the potential for toxigenic or lysogenic conversion, which translate to safety in biocontrol and phage therapy applications. This review article summarizes phage application pipelines at different levels, and addresses specific restrictions and knowledge gaps in the field. Recently developed computational approaches, which are used in phage genome analysis, are critically assessed. We hope that this assessment provides researchers with useful insights for the selection of suitable approaches for phage-related research aims and applications.

Bacteriophage as a potential therapy to control antibiotic-resistant Pseudomonas aeruginosa infection through topical application onto a full-thickness wound in a rat model.

BACKGROUND: Antibiotic-resistant Pseudomonas aeruginosa (P. aeruginosa) is one of the most critical pathogens in wound infections, causing high mortality and morbidity in severe cases. However, bacteriophage therapy is a potential alternative to antibiotics against P. aeruginosa. Therefore, this study aimed to isolate a novel phage targeting P. aeruginosa and examine its efficacy in vitro and in vivo. RESULTS: The morphometric and genomic analyses revealed that ZCPA1 belongs to the Siphoviridae family and could infect 58% of the tested antibiotic-resistant P. aeruginosa clinical isolates. The phage ZCPA1 exhibited thermal stability at 37 °C, and then, it decreased gradually at 50 °C and 60 °C. At the same time, it dropped significantly at 70 °C, and the phage was undetectable at 80 °C. Moreover, the phage ZCPA1 exhibited no significant titer reduction at a wide range of pH values (4-10) with maximum activity at pH 7. In addition, it was stable for 45 min under UV light with one log reduction after 1 h. Also, it displayed significant lytic activity and biofilm elimination against P. aeruginosa by inhibiting bacterial growth in vitro in a dose-dependent pattern with a complete reduction of the bacterial growth at a multiplicity of infection (MOI) of 100. In addition, P. aeruginosa-infected wounds treated with phages displayed 100% wound closure with a high quality of regenerated skin compared to the untreated and gentamicin-treated groups due to the complete elimination of bacterial infection. CONCLUSION: The phage ZCPA1 exhibited high lytic activity against MDR P. aeruginosa planktonic and biofilms. In addition, phage ZCPA1 showed complete wound healing in the rat model. Hence, this research demonstrates the potential of phage therapy as a promising alternative in treating MDR P. aeruginosa.

Isolation and Characterization of Phages Active against Paenibacillus larvae Causing American Foulbrood in Honeybees in Poland.

The aim of this study was the isolation and characterization, including the phage effect on honeybees in laboratory conditions, of phages active against Paenibacillus larvae, the causative agent of American Foulbrood-a highly infective and easily spreading disease occurring in honeybee larva, and subsequently the development of a preparation to prevent and treat this dangerous disease. From the tested material (over 2500 samples) 35 Paenibacillus spp. strains were obtained and used to search for phages. Five phages specific to Paenibacillus were isolated and characterized (ultrastructure, morphology, biological properties, storage stability, and genome sequence). The characteristics were performed to obtain knowledge of their lytic potential and compose the final phage cocktail with high antibacterial potential and intended use of future field application. Preliminary safety studies have also been carried out on healthy bees, which suggest that the phage preparation administered is harmless.

Analysis of Four New Enterococcus faecalis Phages and Modeling of a Hyaluronidase Catalytic Domain from Saphexavirus.

Background: Phage therapy (PT), as a method to treat bacterial infections, needs identification of bacteriophages targeting specific pathogenic host. Enterococcus faecalis, a Gram-positive coccus resident in the human gastrointestinal tract, may become pathogenic in hospitalized patients showing acquired resistance to vancomycin and thus representing a possible target for PT. Materials and Methods: We isolated four phages that infect E. faecalis and characterized them by host range screening, transmission electron microscopy, and genome sequencing. We also identified and three-dimensional modeled a new hyaluronidase enzyme. Results: The four phages belong to Siphoviridae family: three Efquatrovirus (namely vB_EfaS_TV51, vB_EfaS_TV54, and vB_EfaS_TV217) and one Saphexavirus (vB_EfaS_TV16). All of them are compatible with lytic cycle. vB_EfaS_TV16 moreover presents a gene encoding for a hyaluronidase enzyme. Conclusions: The identified phages show features suggesting their useful application in PT, particularly the Saphexavirus that may be of enhanced relevance in PT because of its potential biofilm-digestion capability.

Topically Applied Bacteriophage to Control Multi-Drug Resistant Klebsiella pneumoniae Infected Wound in a Rat Model.

(Background): Multi-drug-resistant Klebsiella pneumoniae (MDR-KP) has steadily grown beyond antibiotic control. Wound infection kills many patients each year, due to the entry of multi-drug resistant (MDR) bacterial pathogens into the skin gaps. However, a bacteriophage (phage) is considered to be a potential antibiotic alternative for treating bacterial infections. This research aims at isolating and characterizing a specific phage and evaluate its topical activity against MDR-KP isolated from infected wounds. (Methods): A lytic phage ZCKP8 was isolated by using a clinical isolate KP/15 as a host strain then characterized. Additionally, phage was assessed for its in vitro host range, temperature, ultraviolet (UV), and pH sensitivity. The therapeutic efficiency of phage suspension and a phage-impeded gel vehicle were assessed in vivo against a K. pneumoniae infected wound on a rat model. (Result): The phage produced a clear plaque and was classified as Siphoviridae. The phage inhibited KP/15 growth in vitro in a dose-dependent pattern and it was found to resist high temperature (˂70 °C) and was primarily active at pH 5; moreover, it showed UV stability for 45 min. Phage-treated K. pneumoniae inoculated wounds showed the highest healing efficiency by lowering the infection. The quality of the regenerated skin was evidenced via histological examination compared to the untreated control group. (Conclusions): This research represents the evidence of effective phage therapy against MDR-KP.

Genome Sequence and Characterization of Five Bacteriophages Infecting Streptomyces Coelicolor and Streptomyces Venezuelae: Alderaan, Coruscant, Dagobah, Endor1 and Endor2.

Streptomyces are well-known antibiotic producers, also characterized by a complex morphological differentiation. Streptomyces, like all bacteria, are confronted with the constant threat of phage predation, which in turn shapes bacterial evolution. However, despite significant sequencing efforts recently, relatively few phages infecting Streptomyces have been characterized compared to other genera. Here, we present the isolation and characterization of five novel Streptomyces phages. All five phages belong to the Siphoviridae family, based on their morphology as determined by transmission electron microscopy. Genome sequencing and life style predictions suggested that four of them were temperate phages, while one had a lytic lifestyle. Moreover, one of the newly sequenced phages shows very little homology to already described phages, highlighting the still largely untapped viral diversity. Altogether, this study expands the number of characterized phages of Streptomyces and sheds light on phage evolution and phage-host dynamics in Streptomyces.

A New High-Throughput Screening Method for Phages: Enabling Crude Isolation and Fast Identification of Diverse Phages with Therapeutic Potential.

Bacteriophage therapy and application of phages for biocontrol necessitate acquisition of suitable phages. The exclusivity of phage-host relations and the risk of phage resistance instigate a need to rapidly isolate and characterize novel phages and continually build sizeable phage libraries. Current methods for phage isolation are both laborious and time consuming, suitable for the isolation of a limited number of phages. The high-throughput screening method for phages upscales and organizes enrichment of phages for fast isolation and identification of potentially hundreds of distinct phages against single hosts. This enables screening of hundreds of samples, in multiple simultaneous setups with varying parameters, increasing the likelihood of isolating multiple distinct phages specific for the given conditions. The efficiency of the method is emphasized by our screening of 200 environmental samples, resulting in the identification of an abundance of unique phage species virulent to Escherichia coli, Salmonella enterica, Enterococcus faecalis, and Pseudomonas aeruginosa.