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12 pages, 2955 KiB

Open AccessArticle

Characterization of a Bacteriophage GEC_vB_Bfr_UZM3 Active against Bacteroides fragilis

by Nata Bakuradze, Maia Merabishvili, Ia Kusradze, Pieter-Jan Ceyssens, Jolien Onsea, Willem-Jan Metsemakers, Nino Grdzelishvili, Guliko Natroshvili, Tamar Tatrishvili, Davit Lazvliashvili, Nunu Mitskevich, Jean-Paul Pirnay and Nina Chanishvili

Viruses 2023, 15(5), 1042; https://doi.org/10.3390/v15051042 - 25 Apr 2023

Cited by 1 | Viewed by 1657

Abstract

Bacteroides fragilis is a commensal gut bacterium that is associated with a number of blood and tissue infections. It has not yet been recognized as one of the drug-resistant human pathogens, but cases of the refractory infections, caused by strains that are not [...] Read more.

Bacteroides fragilis is a commensal gut bacterium that is associated with a number of blood and tissue infections. It has not yet been recognized as one of the drug-resistant human pathogens, but cases of the refractory infections, caused by strains that are not susceptible to the common antibiotic regimes established for B. fragilis, have been more frequently reported. Bacteriophages (phages) were found to be a successful antibacterial alternative to antibiotic therapy in many cases of multidrug-resistant (MDR) bacterial infections. We have characterized the bacteriophage GEC_vB_Bfr_UZM3 (UZM3), which was used for the treatment of a patient with a chronic osteomyelitis caused by a B. fragilis mixed infection. Studied biological and morphological properties of UZM3 showed that it seems to represent a strictly lytic phage belonging to a siphovirus morphotype. It is characterized by high stability at body temperature and in pH environments for about 6 h. Whole genome sequencing analysis of the phage UZM3 showed that it does not harbor any known virulence genes and can be considered as a potential therapeutic phage to be used against B. fragilis infections. Full article

(This article belongs to the Special Issue State-of-the-Art Phage Therapy Development in Europe 2022)

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23 pages, 1100 KiB

Open AccessCommunication

Phage Therapy in Germany—Update 2023

by Christian Willy, Joachim J. Bugert, Annika Y. Classen, Li Deng, Anja Düchting, Justus Gross, Jens A. Hammerl, Imke H. E. Korf, Christian Kühn, Simone Lieberknecht-Jouy, Christine Rohde, Markus Rupp, Maria J. G. T. Vehreschild, Kilian Vogele, Sarah Wienecke, Martin Witzenrath, Silvia Würstle, Holger Ziehr, Karin Moelling and Felix Broecker

Viruses 2023, 15(2), 588; https://doi.org/10.3390/v15020588 - 20 Feb 2023

Cited by 9 | Viewed by 5018

Abstract

Bacteriophage therapy holds promise in addressing the antibiotic-resistance crisis, globally and in Germany. Here, we provide an overview of the current situation (2023) of applied phage therapy and supporting research in Germany. The authors, an interdisciplinary group working on patient-focused bacteriophage research, addressed [...] Read more.

Bacteriophage therapy holds promise in addressing the antibiotic-resistance crisis, globally and in Germany. Here, we provide an overview of the current situation (2023) of applied phage therapy and supporting research in Germany. The authors, an interdisciplinary group working on patient-focused bacteriophage research, addressed phage production, phage banks, susceptibility testing, clinical application, ongoing translational research, the regulatory situation, and the network structure in Germany. They identified critical shortcomings including the lack of clinical trials, a paucity of appropriate regulation and a shortage of phages for clinical use. Phage therapy is currently being applied to a limited number of patients as individual treatment trials. There is presently only one site in Germany for large-scale good-manufacturing-practice (GMP) phage production, and one clinic carrying out permission-free production of medicinal products. Several phage banks exist, but due to varying institutional policies, exchange among them is limited. The number of phage research projects has remarkably increased in recent years, some of which are part of structured networks. There is a demand for the expansion of production capacities with defined quality standards, a structured registry of all treated patients and clear therapeutic guidelines. Furthermore, the medical field is still poorly informed about phage therapy. The current status of non-approval, however, may also be regarded as advantageous, as insufficiently restricted use of phage therapy without adequate scientific evidence for effectiveness and safety must be prevented. In close coordination with the regulatory authorities, it seems sensible to first allow some centers to treat patients following the Belgian model. There is an urgent need for targeted networking and funding, particularly of translational research, to help advance the clinical application of phages. Full article

(This article belongs to the Special Issue State-of-the-Art Phage Therapy Development in Europe 2022)

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12 pages, 256 KiB

Open AccessArticle

More’s the Same—Multiple Hosts Do Not Select for Broader Host Range Phages

by Jupiter Myers, Joshua Davis II, Megan Lollo, Gabriella Hudec and Paul Hyman

Viruses 2023, 15(2), 518; https://doi.org/10.3390/v15020518 - 13 Feb 2023

Cited by 5 | Viewed by 2720

Abstract

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. [...] Read more.

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. Full article

(This article belongs to the Special Issue State-of-the-Art Phage Therapy Development in Europe 2022)

19 pages, 1110 KiB

Open AccessArticle

Short-Term Outcomes of Phage-Antibiotic Combination Treatment in Adult Patients with Periprosthetic Hip Joint Infection

by Eugeny Fedorov, Alexander Samokhin, Yulia Kozlova, Svetlana Kretien, Taalai Sheraliev, Vera Morozova, Nina Tikunova, Alexey Kiselev and Vitaliy Pavlov

Viruses 2023, 15(2), 499; https://doi.org/10.3390/v15020499 - 10 Feb 2023

Cited by 8 | Viewed by 1899

Abstract

Implant-associated infections are the most costly problem in modern orthopedics due to the continued increase in the occurrence of antibiotic-resistant bacterial strains that requires the development of new effective antimicrobials. A non-randomized, prospective, open-label, with historical control study on the use of combined [...] Read more.

Implant-associated infections are the most costly problem in modern orthopedics due to the continued increase in the occurrence of antibiotic-resistant bacterial strains that requires the development of new effective antimicrobials. A non-randomized, prospective, open-label, with historical control study on the use of combined phage/antibiotic therapy of periprosthetic joint infection (PJI) was carried out. Forty-five adult patients with deep PJI of the hip joint were involved in the study, with a 12-month follow-up after one-stage revision surgery. Patients from a prospective study group (SG, n = 23) were treated with specific phage preparation and etiotropic antibiotics, whereas patients from a retrospective comparator group (CG, n = 22) received antibiotics only. The rate of PJI relapses in the SG was eight times less than that in the CG: one case (4.5%) versus eight cases (36.4%), p = 0.021. The response rate to treatment was 95.5% (95% confidence interval (CI) = 0.7511–0.9976) in the SG and only 63.6% (95% CI = 0.4083–0.8198) in the CG. The odds ratio for PJI relapse in patients of the SG was 0.083 (95% CI = 0.009–0.742), which was almost 12 times lower than that in the CG. The obtained results support the efficacy of the combined phage-antibiotic treatment of PJI. Full article

(This article belongs to the Special Issue State-of-the-Art Phage Therapy Development in Europe 2022)

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21 pages, 3419 KiB

Open AccessArticle

Repetitive Exposure to Bacteriophage Cocktails against Pseudomonas aeruginosa or Escherichia coli Provokes Marginal Humoral Immunity in Naïve Mice

by Chantal Weissfuss, Sandra-Maria Wienhold, Magdalena Bürkle, Baptiste Gaborieau, Judith Bushe, Ulrike Behrendt, Romina Bischoff, Imke H. E. Korf, Sarah Wienecke, Antonia Dannheim, Holger Ziehr, Christine Rohde, Achim D. Gruber, Jean-Damien Ricard, Laurent Debarbieux, Martin Witzenrath and Geraldine Nouailles

Viruses 2023, 15(2), 387; https://doi.org/10.3390/v15020387 - 29 Jan 2023

Cited by 2 | Viewed by 2223

Abstract

Phage therapy of ventilator-associated pneumonia (VAP) is of great interest due to the rising incidence of multidrug-resistant bacterial pathogens. However, natural or therapy-induced immunity against therapeutic phages remains a potential concern. In this study, we investigated the innate and adaptive immune responses to [...] Read more.

Phage therapy of ventilator-associated pneumonia (VAP) is of great interest due to the rising incidence of multidrug-resistant bacterial pathogens. However, natural or therapy-induced immunity against therapeutic phages remains a potential concern. In this study, we investigated the innate and adaptive immune responses to two different phage cocktails targeting either Pseudomonas aeruginosa or Escherichia coli—two VAP-associated pathogens—in naïve mice without the confounding effects of a bacterial infection. Active or UV-inactivated phage cocktails or buffers were injected intraperitoneally daily for 7 days in C57BL/6J wild-type mice. Blood cell analysis, flow cytometry analysis, assessment of phage distribution and histopathological analysis of spleens were performed at 6 h, 10 days and 21 days after treatment start. Phages reached the lungs and although the phage cocktails were slightly immunogenic, phage injections were well tolerated without obvious adverse effects. No signs of activation of innate or adaptive immune cells were observed; however, both active phage cocktails elicited a minimal humoral response with secretion of phage-specific antibodies. Our findings show that even repetitive injections lead only to a minimal innate and adaptive immune response in naïve mice and suggest that systemic phage treatment is thus potentially suitable for treating bacterial lung infections. Full article

(This article belongs to the Special Issue State-of-the-Art Phage Therapy Development in Europe 2022)

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17 pages, 2265 KiB

Open AccessArticle

Chimeric Peptidoglycan Hydrolases Kill Staphylococcal Mastitis Isolates in Raw Milk and within Bovine Mammary Gland Epithelial Cells

by Anja P. Keller, Shera Ly, Steven Daetwyler, Fritz Eichenseher, Martin J. Loessner and Mathias Schmelcher

Viruses 2022, 14(12), 2801; https://doi.org/10.3390/v14122801 - 15 Dec 2022

Cited by 2 | Viewed by 1416

Abstract

Staphylococcus aureus is a major causative agent of bovine mastitis, a disease considered one of the most economically devastating in the dairy sector. Considering the increasing prevalence of antibiotic-resistant strains, novel therapeutic approaches efficiently targeting extra- and intracellular bacteria and featuring high activity [...] Read more.

Staphylococcus aureus is a major causative agent of bovine mastitis, a disease considered one of the most economically devastating in the dairy sector. Considering the increasing prevalence of antibiotic-resistant strains, novel therapeutic approaches efficiently targeting extra- and intracellular bacteria and featuring high activity in the presence of raw milk components are needed. Here, we have screened a library of eighty peptidoglycan hydrolases (PGHs) for high activity against S. aureus in raw bovine milk, twelve of which were selected for further characterization and comparison in time-kill assays. The bacteriocins lysostaphin and ALE-1, and the chimeric PGH M23LST(L)_SH3b2638 reduced bacterial numbers in raw milk to the detection limit within 10 min. Three CHAP-based PGHs (CHAPGH15_SH3bAle1, CHAPK_SH3bLST_H, CHAPH5_LST_H) showed gradually improving activity with increasing dilution of the raw milk. Furthermore, we demonstrated synergistic activity of CHAPGH15_SH3bAle1 and LST when used in combination. Finally, modification of four PGHs (LST, M23LST(L)_SH3b2638, CHAPK_SH3bLST, CHAPGH15_SH3bAle1) with the cell-penetrating peptide TAT significantly enhanced the eradication of intracellular S. aureus in bovine mammary alveolar cells compared to the unmodified parentals in a concentration-dependent manner. Full article

(This article belongs to the Special Issue State-of-the-Art Phage Therapy Development in Europe 2022)

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10 pages, 1127 KiB

Open AccessArticle

Subtherapeutic Doses of Vancomycin Synergize with Bacteriophages for Treatment of Experimental Methicillin-Resistant Staphylococcus aureus Infective Endocarditis

by Jonathan Save, Yok-Ai Que, José Entenza and Grégory Resch

Viruses 2022, 14(8), 1792; https://doi.org/10.3390/v14081792 - 16 Aug 2022

Cited by 4 | Viewed by 1577

Abstract

Background. Recurrent therapeutic failures reported for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infective endocarditis (IE) with vancomycin may be due to poor bactericidal activity. Alternative antibacterial approaches using bacteriophages may overcome this limitation. Objectives. An experimental rat model of MRSA IE (EE) [...] Read more.

Background. Recurrent therapeutic failures reported for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infective endocarditis (IE) with vancomycin may be due to poor bactericidal activity. Alternative antibacterial approaches using bacteriophages may overcome this limitation. Objectives. An experimental rat model of MRSA IE (EE) was used to examine the efficacy of vancomycin combined with a 1:1 bacteriophage (phage) cocktail composed of Herelleviridae vB_SauH_2002 and Routreeviridae 66. Methods. Six hours after inoculation with ca. 5 log₁₀ colony forming units (CFU) of MRSA strain AW7, animals were treated with either: (i) saline, (ii) an equimolar two-phage cocktail (bolus of 1 mL followed by a 0.3 mL/h continuous infusion of 10 log₁₀ plaque forming units (PFU)/mL phage suspension), (iii) vancomycin (at a dose mimicking the kinetics in humans of 0.5 g b.i.d.), or (iv) a combination of both. Bacterial loads in vegetations, and phage loads in vegetations, liver, kidney, spleen, and blood, were measured outcomes. Results. Phage cocktail alone was unable to control the growth of strain AW7 in cardiac vegetations. However, when combined with subtherapeutic doses of vancomycin, a statistically significant decrease of ∆4.05 ± 0.94 log₁₀ CFU/g at 24 h compared to placebo was detected (p < 0.001). The administration of vancomycin was found to significantly impact on the local concentrations of phages in the vegetations and in the organs examined. Conclusions. Lytic bacteriophages as an adjunct treatment to the standard of care antibiotics could potentially improve the management of MRSA IE. Further studies are needed to investigate the impact of antibiotics on phage replication in vivo. Full article

(This article belongs to the Special Issue State-of-the-Art Phage Therapy Development in Europe 2022)

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14 pages, 2176 KiB

Open AccessArticle

An Optimized Checkerboard Method for Phage-Antibiotic Synergy Detection

by Isidora Nikolic, Darija Vukovic, Damir Gavric, Jelena Cvetanovic, Verica Aleksic Sabo, Sonja Gostimirovic, Jelena Narancic and Petar Knezevic

Viruses 2022, 14(7), 1542; https://doi.org/10.3390/v14071542 - 14 Jul 2022

Cited by 8 | Viewed by 4961

Abstract

Phage-antibiotic synergy is a promising therapeutic strategy, but there is no reliable method for synergism estimation. Although the time-kill curve assay is a gold standard, the method is not appropriate for fast and extensive screening of the synergy. The aim is to optimize [...] Read more.

Phage-antibiotic synergy is a promising therapeutic strategy, but there is no reliable method for synergism estimation. Although the time-kill curve assay is a gold standard, the method is not appropriate for fast and extensive screening of the synergy. The aim is to optimize the checkerboard method to determine phage-chemical agent interactions, to check its applicability by the time-kill curve method, and to examine whether the synergy can be obtained with both simultaneous and successive applications of these agents. In addition, the aim is to determine interactions of the Pseudomonas phage JG024 with ciprofloxacin, gentamicin, or ceftriaxone, as well as the Staphylococcus phage MSA6 and SES43300 with ciprofloxacin, gentamicin, and oxacillin. The results show that the optimized checkerboard method is reliable and that results correspond to those obtained by the time-kill curve. The synergy is detected with the phage JG024 and ciprofloxacin or ceftriaxone against Pseudomonas aeruginosa, and the phage SES43300 with ciprofloxacin against MRSA. The synergy was obtained after simultaneous applications, and in the case of P. aeruginosa, after application of the second agent with delay of one hour, indicating that simultaneous application is the best mode of synergy exploitation for therapy. The checkerboard method can be used for thorough clinical studies on synergy and in the future for personalized therapy when infections are caused by multiple resistant bacteria. Full article

(This article belongs to the Special Issue State-of-the-Art Phage Therapy Development in Europe 2022)

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21 pages, 2163 KiB

Open AccessArticle

Filamentous Pseudomonas Phage Pf4 in the Context of Therapy-Inducibility, Infectivity, Lysogenic Conversion, and Potential Application

by Damir Gavric and Petar Knezevic

Viruses 2022, 14(6), 1261; https://doi.org/10.3390/v14061261 - 10 Jun 2022

Cited by 9 | Viewed by 2554

Abstract

More than 20% of all Pseudomonas aeruginosa are infected with Pf4-related filamentous phage and although their role in virulence of P. aeruginosa strain PAO1 is well documented, its properties related to therapy are not elucidated in detail. The aim of this study was [...] Read more.

More than 20% of all Pseudomonas aeruginosa are infected with Pf4-related filamentous phage and although their role in virulence of P. aeruginosa strain PAO1 is well documented, its properties related to therapy are not elucidated in detail. The aim of this study was to determine how phage and antibiotic therapy induce Pf4, whether the released virions can infect other strains and how the phage influences the phenotype of new hosts. The subinhibitory concentrations of ciprofloxacin and mitomycin C increased Pf4 production for more than 50% during the first and sixth hour of exposure, respectively, while mutants appearing after infection with obligatory lytic phage at low MOI produced Pf4 more than four times after 12–24 h of treatment. This indicates that production of Pf4 is enhanced during therapy with these agents. The released virions can infect new P. aeruginosa strains, as confirmed for models UCBPP-PA14 (PA14) and LESB58, existing both episomally and in a form of a prophage, as confirmed by PCR, RFLP, and sequencing. The differences in properties of Pf4-infected, and uninfected PA14 and LESB58 strains were obvious, as infection with Pf4 significantly decreased cell autoaggregation, pyoverdine, and pyocyanin production, while significantly increased swimming motility and biofilm production in both strains. In addition, in strain PA14, Pf4 increased cell surface hydrophobicity and small colony variants’ appearance, but also decreased twitching and swarming motility. This indicates that released Pf4 during therapy can infect new strains and cause lysogenic conversion. The infection with Pf4 increased LESB58 sensitivity to ciprofloxacin, gentamicin, ceftazidime, tetracycline, and streptomycin, and PA14 to ciprofloxacin and ceftazidime. Moreover, the Pf4-infected LESB58 was re-sensitized to ceftazidime and tetracycline, with changes from resistant to intermediate resistant and sensitive, respectively. The obtained results open a new field in phage therapy—treatment with selected filamentous phages in order to re-sensitize pathogenic bacteria to certain antibiotics. However, this approach should be considered with precautions, taking into account potential lysogenic conversion. Full article

(This article belongs to the Special Issue State-of-the-Art Phage Therapy Development in Europe 2022)

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20 pages, 1758 KiB

Open AccessArticle

A Thorough Synthesis of Phage Therapy Unit Activity in Poland—Its History, Milestones and International Recognition

by Maciej Żaczek, Andrzej Górski, Beata Weber-Dąbrowska, Sławomir Letkiewicz, Wojciech Fortuna, Paweł Rogóż, Edyta Pasternak and Ryszard Międzybrodzki

Viruses 2022, 14(6), 1170; https://doi.org/10.3390/v14061170 - 28 May 2022

Cited by 9 | Viewed by 2378

Abstract

The year 2020 marked 15 years of the Phage Therapy Unit in Poland, the inception of which took place just one year after Poland’s accession to the European Union (2004). At first sight, it is hard to find any connection between these two [...] Read more.

The year 2020 marked 15 years of the Phage Therapy Unit in Poland, the inception of which took place just one year after Poland’s accession to the European Union (2004). At first sight, it is hard to find any connection between these two events, but in fact joining the European Union entailed the need to adapt the regulatory provisions concerning experimental treatment in humans to those that were in force in the European Union. These changes were a solid foundation for the first phage therapy center in the European Union to start its activity. As the number of centers conducting phage therapy in Europe and in the world constantly and rapidly grows, we want to grasp the opportunity to take a closer look at the over 15-year operation of our site by analyzing its origins, legal aspects at the local and international levels and the impressive number and diversity of cases that have been investigated and treated during this time. This article is a continuation of our work published in 2020 summarizing a 100-year history of the development of phage research in Poland. Full article

(This article belongs to the Special Issue State-of-the-Art Phage Therapy Development in Europe 2022)

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Review

Jump to: Research

23 pages, 2842 KiB

Open AccessReview

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

by Tea Glonti and Jean-Paul Pirnay

Viruses 2022, 14(7), 1490; https://doi.org/10.3390/v14071490 - 07 Jul 2022

Cited by 32 | Viewed by 5751

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue State-of-the-Art Phage Therapy Development in Europe 2022)

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