Isolation and Characterization of the Novel Bacteriophage AXL3 against Stenotrophomonas maltophilia

The rapid increase in the number of worldwide human infections caused by the extremely antibiotic resistant bacterial pathogen Stenotrophomonas maltophilia is cause for concern. An alternative treatment solution in the post-antibiotic era is phage therapy, the use of bacteriophages to selectively kill bacterial pathogens. In this study, the novel bacteriophage AXL3 (vB_SmaS-AXL_3) was isolated from soil and characterized. Host range analysis using a panel of 29 clinical S. maltophilia isolates shows successful infection of five isolates and electron microscopy indicates that AXL3 is a member of the Siphoviridae family. Complete genome sequencing and analysis reveals a 47.5 kb genome predicted to encode 65 proteins. Functionality testing suggests AXL3 is a virulent phage and results show that AXL3 uses the type IV pilus, a virulence factor on the cell surface, as its receptor across its host range. This research identifies a novel virulent phage and characterization suggests that AXL3 is a promising phage therapy candidate, with future research examining modification through genetic engineering to broaden its host range.

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The Potential of Phage Therapy against the Emerging Opportunistic Pathogen Stenotrophomonas maltophilia

Viruses ◽

10.3390/v13061057 ◽

2021 ◽

Vol 13 (6) ◽

pp. 1057

Author(s):

Jaclyn G. McCutcheon ◽

Jonathan J. Dennis

Keyword(s):

Antimicrobial Resistance ◽

Virulence Factors ◽

Stenotrophomonas Maltophilia ◽

Phage Therapy ◽

Multidrug Resistant ◽

Future Research ◽

Isolation And Characterization ◽

Alternative Treatment Option ◽

Antimicrobial Resistance Genes

The isolation and characterization of bacteriophages for the treatment of infections caused by the multidrug resistant pathogen Stenotrophomonas maltophilia is imperative as nosocomial and community-acquired infections are rapidly increasing in prevalence. This increase is largely due to the numerous virulence factors and antimicrobial resistance genes encoded by this bacterium. Research on S. maltophilia phages to date has focused on the isolation and in vitro characterization of novel phages, often including genomic characterization, from the environment or by induction from bacterial strains. This review summarizes the clinical significance, virulence factors, and antimicrobial resistance mechanisms of S. maltophilia, as well as all phages isolated and characterized to date and strategies for their use. We further address the limited in vivo phage therapy studies conducted against this bacterium and discuss the future research needed to spearhead phages as an alternative treatment option against multidrug resistant S. maltophilia.

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Efficiency of Phage φ6 for Biocontrol of Pseudomonas syringae pv. syringae: An in Vitro Preliminary Study

Microorganisms ◽

10.3390/microorganisms7090286 ◽

2019 ◽

Vol 7 (9) ◽

pp. 286 ◽

Cited By ~ 11

Author(s):

Larindja A. M. Pinheiro ◽

Carla Pereira ◽

Carolina Frazão ◽

Victor M. Balcão ◽

Adelaide Almeida

Keyword(s):

Solar Radiation ◽

Host Range ◽

Pseudomonas Syringae ◽

Bacterial Resistance ◽

Phage Therapy ◽

Bacterial Species ◽

Fruit Trees ◽

Range Analysis ◽

Maximum Reduction

Pseudomonas syringae is a plant-associated bacterial species that has been divided into more than 60 pathovars, with the Pseudomonas syringae pv. syringae being the main causative agent of diseases in a wide variety of fruit trees. The most common treatments for biocontrol of P. syringae pv. syringae infections has involved copper derivatives and/or antibiotics. However, these treatments should be avoided due to their high toxicity to the environment and promotion of bacterial resistance. Therefore, it is essential to search for new approaches for controlling P. syringae pv. syringae. Phage therapy can be a useful alternative tool to the conventional treatments to control P. syringae pv. syringae infections in plants. In the present study, the efficacy of bacteriophage (or phage) φ6 (a commercially available phage) was evaluated in the control of P. syringae pv. syringae. As the plants are exposed to the natural variability of physical and chemical parameters, the influence of pH, temperature, solar radiation and UV-B irradiation on phage φ6 viability was also evaluated in order to develop an effective phage therapy protocol. The host range analysis revealed that the phage, besides its host (P. syringae pv. syringae), also infects the Pseudomonas syringae pv. actinidiae CRA-FRU 12.54 and P. syringae pv. actinidiae CRA-FRU 14.10 strains, not infecting strains from the other tested species. Both multiplicities of infection (MOIs) tested, 1 and 100, were effective to inactivate the bacterium, but the MOI 1 (maximum reduction of 3.9 log CFU/mL) was more effective than MOI 100 (maximum reduction of 2.6 log CFU/mL). The viability of phage φ6 was mostly affected by exposure to UV-B irradiation (decrease of 7.3 log PFU/mL after 8 h), exposure to solar radiation (maximum reduction of 2.1 PFU/mL after 6 h), and high temperatures (decrease of 8.5 PFU/mL after 6 days at 37 °C, but a decrease of only 2.0 log PFU/mL after 67 days at 15 °C and 25 °C). The host range, high bacterial control and low rates of development of phage-resistant bacterial clones (1.20 × 10−3) suggest that this phage can be used to control P. syringae pv. syringae infections in plants, but also to control infections by P. syringae pv. actinidiae, the causal agent of bacterial canker of kiwifruit. Although the stability of phage φ6 was affected by UV-B and solar radiation, this can be overcome by the application of phage suspensions at the end of the day or at night.

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Isolation and Characterization of Novel Giant Stenotrophomonas maltophilia Phage φSMA5

Applied and Environmental Microbiology ◽

10.1128/aem.71.3.1387-1393.2005 ◽

2005 ◽

Vol 71 (3) ◽

pp. 1387-1393 ◽

Cited By ~ 68

Author(s):

Hsiao-Chuan Chang ◽

Chiy-Rong Chen ◽

Juey-Wen Lin ◽

Gwan-Han Shen ◽

Kai-Ming Chang ◽

...

Keyword(s):

Gel Electrophoresis ◽

Stenotrophomonas Maltophilia ◽

Phage Therapy ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Restriction Enzymes ◽

Clinical Samples ◽

Opportunistic Bacteria ◽

Isolation And Characterization ◽

Therapy Strategy

ABSTRACT Stenotrophomonas maltophilia is one of the most prevalent opportunistic bacteria causing nosocomial infections. It has become problematic because most of the isolates are resistant to multiple antibiotics, and therefore, development of phage therapy has attracted strong attention. In this study, eight S. maltophilia phages were isolated from clinical samples including patient specimens, catheter-related devices, and wastewater. These phages can be divided into four distinct groups based on host range and digestibility of the phage DNAs with different restriction endonucleases. One of them, designated φSMA5, was further characterized. Electron microscopy showed it resembled Myoviridae, with an isometric head (90 nm in diameter), a tail (90 nm long), a baseplate (25 nm wide), and short tail fibers. The φSMA5 double-stranded DNA, refractory to digestion by most restriction enzymes, was tested and estimated to be 250 kb by pulsed-field gel electrophoresis. This genome size is second to that of the largest phage, φKZ of Pseudomonas aeruginosa. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 25 virion proteins were visualized. N-terminal sequencing of four of them suggested that each of them might have had its N terminus cleaved off. Among the 87 S. maltophilia strains collected in this study, only 61 were susceptible to φSMA5, indicating that more phages are needed toward a phage therapy strategy. Since literature search yielded no information about S. maltophilia phages, φSMA5 appears to be the first reported.

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Alterations in gp37 Expand the Host Range of a T4-Like Phage

Applied and Environmental Microbiology ◽

10.1128/aem.01576-17 ◽

2017 ◽

Vol 83 (23) ◽

Cited By ~ 13

Author(s):

Mianmian Chen ◽

Lei Zhang ◽

Sheikheldin Adam Abdelgader ◽

Li Yu ◽

Juntian Xu ◽

...

Keyword(s):

Host Range ◽

First Generation ◽

Phage Therapy ◽

Genetic Manipulation ◽

Homologous Sequence ◽

Sequencing Analysis ◽

The Novel ◽

Direct Expansion ◽

Temperature Range ◽

Sequence Region

ABSTRACT The use of phages as antibacterial agents is limited by their generally narrow host ranges. The aim of this study was to make a T4-like phage, WG01, obtain the host range of another T4-like phage, QL01, by replacing its host-determinant gene region with that of QL01. This process triggered a direct expansion of the WG01 host range. The offspring of WG01 obtained the host ranges of both QL01 and WG01, as well as the ability to infect eight additional host bacteria in comparison to the wild-type strains. WQD had the widest host range; therefore, the corresponding fragments, named QD, could be used for constructing a homologous sequence library. Moreover, after a sequencing analysis of gene 37, we identified two different mechanisms responsible for the expanded host range: (i) the first generation of WG01 formed chimeras without mutations, and (ii) the second generation of WG01 mutants formed from the chimeras. The expansion of the host range indicated that regions other than the C-terminal region may indirectly change the receptor specificity by altering the supportive capacity of the binding site. Additionally, we also found the novel means by which subsequent generations expanded their host ranges, namely, by exchanging gene 37 to acquire a wider temperature range for lysis. The method developed in this work offers a quick way to change or expand the host range of a phage. Future clinical applications for screening phages against a given clinical isolate could be achieved after acquiring more suitable homologous sequences. IMPORTANCE T4-like phages have been established as safe in numerous phage therapy applications. The primary drawbacks to the use of phages as therapeutic agents include their highly specific host ranges. Thus, changing or expanding the host range of T4-like phages is beneficial for selecting phages for phage therapy. In this study, the host range of the T4-like phage WG01 was expanded using genetic manipulation. The WG01 derivatives acquired a novel means of expanding their host ranges by acquiring a wider temperature range for lysis. A region was located that had the potential to be used as a sequence region for homologous sequence recombination.

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Isolation and Characterization of a Novel Phage SaGU1 that Infects Staphylococcus aureus Clinical Isolates from Patients with Atopic Dermatitis

Current Microbiology ◽

10.1007/s00284-021-02395-y ◽

2021 ◽

Vol 78 (4) ◽

pp. 1267-1276

Author(s):

Yuzuki Shimamori ◽

Ajeng K. Pramono ◽

Tomoe Kitao ◽

Tohru Suzuki ◽

Shin-ichi Aizawa ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Atopic Dermatitis ◽

Host Range ◽

Human Skin ◽

Phage Therapy ◽

Gc Content ◽

Clinical Isolates ◽

Trna Genes ◽

Potential Candidate ◽

Isolation And Characterization

AbstractThe bacterium Staphylococcus aureus, which colonizes healthy human skin, may cause diseases, such as atopic dermatitis (AD). Treatment for such AD cases involves antibiotic use; however, alternate treatments are preferred owing to the development of antimicrobial resistance. This study aimed to characterize the novel bacteriophage SaGU1 as a potential agent for phage therapy to treat S. aureus infections. SaGU1 that infects S. aureus strains previously isolated from the skin of patients with AD was screened from sewage samples in Gifu, Japan. Its genome was sequenced and analyzed using bioinformatics tools, and the morphology, lytic activity, stability, and host range of the phage were determined. The SaGU1 genome was 140,909 bp with an average GC content of 30.2%. The viral chromosome contained 225 putative protein-coding genes and four tRNA genes, carrying neither toxic nor antibiotic resistance genes. Electron microscopy analysis revealed that SaGU1 belongs to the Myoviridae family. Stability tests showed that SaGU1 was heat-stable under physiological and acidic conditions. Host range testing revealed that SaGU1 can infect a broad range of S. aureus clinical isolates present on the skin of AD patients, whereas it did not kill strains of Staphylococcus epidermidis, which are symbiotic resident bacteria on human skin. Hence, our data suggest that SaGU1 is a potential candidate for developing a phage therapy to treat AD caused by pathogenic S. aureus.

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Isolation and characterization of a novel phage SaGU1 that infects Staphylococcus aureus clinical isolates from patients with atopic dermatitis

10.1101/2020.10.24.353185 ◽

2020 ◽

Cited By ~ 1

Author(s):

Yuzuki Shimamori ◽

Ajeng K. Pramono ◽

Tomoe Kitao ◽

Tohru Suzuki ◽

Shin-ichi Aizawa ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Atopic Dermatitis ◽

Host Range ◽

Human Skin ◽

Phage Therapy ◽

Gc Content ◽

Clinical Isolates ◽

Trna Genes ◽

Potential Candidate ◽

Isolation And Characterization

AbstractThe bacterium Staphylococcus aureus, which grows on healthy human skin, may cause diseases such as atopic dermatitis (AD). Treatment for such AD cases involves antibiotic use; however, alternate treatments are preferred owing to the development of antimicrobial resistance. This study aimed to characterize the novel bacteriophage SaGU1 as a potential agent for phage therapy to treat S. aureus infections. SaGU1 that infects S. aureus strains previously isolated from the skin of patients with AD was screened from sewage samples in Gifu, Japan. Its genome was sequenced and analyzed using bioinformatics tools, and the morphology, lytic activity, stability, and host range of the phage were determined. The SaGU1 genome consisted of 140,909 bp with an average GC content of 30.2%. The viral chromosome contained putative 225 protein-coding genes and four tRNA genes, carrying neither toxic nor antibiotic resistance genes. Electron microscopy analysis revealed that SaGU1 belongs to the Myoviridae family. Stability tests showed that SaGU1 was heat-stable under physiological and acidic conditions. Host-range testing revealed that SaGU1 could infect a broad range of S. aureus clinical isolates present on the skin of patients with AD, whereas it did not kill strains of Staphylococcus epidermidis, which are symbiotic bacteria in the human skin microbiota. Our data suggest that SaGU1 is a potential candidate for developing a phage therapy to treat AD caused by pathogenic S. aureus.

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Phages for Phage Therapy: Isolation, Characterization, and Host Range Breadth

Pharmaceuticals ◽

10.3390/ph12010035 ◽

2019 ◽

Vol 12 (1) ◽

pp. 35 ◽

Cited By ~ 49

Author(s):

Paul Hyman

Keyword(s):

Host Range ◽

Phage Therapy ◽

Bacterial Pathogen ◽

Broad Host Range ◽

Toxin Genes

For a bacteriophage to be useful for phage therapy it must be both isolated from the environment and shown to have certain characteristics beyond just killing strains of the target bacterial pathogen. These include desirable characteristics such as a relatively broad host range and a lack of other characteristics such as carrying toxin genes and the ability to form a lysogen. While phages are commonly isolated first and subsequently characterized, it is possible to alter isolation procedures to bias the isolation toward phages with desirable characteristics. Some of these variations are regularly used by some groups while others have only been shown in a few publications. In this review I will describe (1) isolation procedures and variations that are designed to isolate phages with broader host ranges, (2) characterization procedures used to show that a phage may have utility in phage therapy, including some of the limits of such characterization, and (3) results of a survey and discussion with phage researchers in industry and academia on the practice of characterization of phages.

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Plant innate immunity in rice: a defense against pathogen infection

National Science Review ◽

10.1093/nsr/nww015 ◽

2016 ◽

Vol 3 (3) ◽

pp. 295-308 ◽

Cited By ~ 20

Author(s):

Wende Liu ◽

Guo-Liang Wang

Keyword(s):

Fungal Pathogen ◽

Bacterial Pathogen ◽

Extensive Study ◽

Future Research ◽

The Novel ◽

Receptor Complexes ◽

The Past ◽

Molecular Pattern ◽

Effector Triggered Immunity ◽

Microbe Interactions

Abstract A large number of pathogenic microorganisms cause rice diseases that lead to enormous yield losses worldwide. Such losses are important because rice is a staple food for more than half of the world's population. Over the past two decades, the extensive study of the molecular interactions between rice and the fungal pathogen Magnaporthe oryzae and between rice and the bacterial pathogen Xanthomonas oryzae pv. oryzae has made rice a model for investigating plant–microbe interactions of monocotyledons. Impressive progress has been recently achieved in understanding the molecular basis of rice pathogen-associated molecular pattern-immunity and effector-triggered immunity. Here, we briefly summarize these recent advances, emphasizing the diverse functions of the structurally conserved fungal effectors, the regulatory mechanisms of the immune receptor complexes, and the novel strategies for breeding disease resistance. We also discuss future research challenges.

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1615. Isolation of Lytic Bacteriophages with Broad Host Range Activity Against Pseudomonas aeruginosa Strains Isolated from Respiratory Samples from Cystic Fibrosis Patients Intended for Therapeutic Application

Open Forum Infectious Diseases ◽

10.1093/ofid/ofaa439.1795 ◽

2020 ◽

Vol 7 (Supplement_1) ◽

pp. S801-S801

Author(s):

Jose Alexander ◽

Daniel Navas ◽

Marly Flowers ◽

Angela Charles ◽

Amy Carr

Keyword(s):

Cystic Fibrosis ◽

Host Range ◽

Phage Therapy ◽

Clinical Isolates ◽

Clinical Samples ◽

Plaque Morphology ◽

Broad Host Range ◽

Chronic Infections ◽

Clinically Significant ◽

Host Target

Abstract Background With the rise of the antimicrobial resistance between different genera and species of bacteria, Phage Therapy is becoming a more realistic and accessible option for patients with limited or no antimicrobial options. Being able to have rapid access to a collection of clinical active phages is key for rapid implementation of phage therapy. The Microbiology Department at AdventHealth Orlando is performing routine screening of environmental and patient samples for isolation of phages against non-fermenting Gram negative bacteria to develop a Phage Bank. Methods Protocols for phage isolation from environmental sources such as lakes, rivers and sewers and clinical samples were developed. A series of respiratory, throat, stool and urine samples were processed following an internal protocol that includes centrifugation, filtration and enrichment. Clinical samples were centrifugated for 10 minutes, filtered using 0.45µm centrifugation filters, seeded with targeted host bacteria (clinical isolates) and incubated at 35°C for 24 hours. The enriched samples were centrifugated and filtered for a final phage enriched solution. Screening and isolation were performed using the Gracia method over trypticase soybean agar (TSA) for plaque morphology and quantification. Host range screening of other clinical isolates of P. aeruginosa was performed using the new isolated and purified phages. Results 4 lytic phages against clinical strains of P. aeruginosa from patient with diagnosis of cystic fibrosis (CF), were isolated and purified from 4 different respiratory samples, including sputum and bronchial alveolar lavage. All phages showed phenotypical characteristics of lytic activity. 1 phage was active against 4 strains of P. aeruginosa, 1 phage was active against 2 strains of P. aeruginosa and the remaining 2 phages were active only against the initial host target strain. Conclusion With this study we demonstrated the potential use of clinical samples as source for isolating active bacteriophages against clinically significant bacteria strains. Clinical samples from vulnerable population of patients with chronic infections are part of our routine “phage-hunting” process to stock and grow our Phage Bank project for future clinical use. Disclosures All Authors: No reported disclosures

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Isolation and Characterization of Salmonella Jumbo-Phage pSal-SNUABM-04

Viruses ◽

10.3390/v13010027 ◽

2020 ◽

Vol 13 (1) ◽

pp. 27

Author(s):

Jun Kwon ◽

Sang Guen Kim ◽

Hyoun Joong Kim ◽

Sib Sankar Giri ◽

Sang Wha Kim ◽

...

Keyword(s):

Morphological Traits ◽

Open Reading Frames ◽

Genome Comparison ◽

The Novel ◽

Promising Alternative ◽

Isolation And Characterization ◽

Global Issue ◽

Reading Frames ◽

Predicted Function

The increasing emergence of antimicrobial resistance has become a global issue. Therefore, many researchers have attempted to develop alternative antibiotics. One promising alternative is bacteriophage. In this study, we focused on a jumbo-phage infecting Salmonella isolated from exotic pet markets. Using a Salmonella strain isolated from reptiles as a host, we isolated and characterized the novel jumbo-bacteriophage pSal-SNUABM-04. This phage was investigated in terms of its morphology, host infectivity, growth and lysis kinetics, and genome. The phage was classified as Myoviridae based on its morphological traits and showed a comparatively wide host range. The lysis efficacy test showed that the phage can inhibit bacterial growth in the planktonic state. Genetic analysis revealed that the phage possesses a 239,626-base pair genome with 280 putative open reading frames, 76 of which have a predicted function and 195 of which have none. By genome comparison with other jumbo phages, the phage was designated as a novel member of Machinavirus composed of Erwnina phages.

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