Therapeutic Phage Research Articles

Klebsiella pneumoniae Phage M198 and Its Therapeutic Potential

The rapid worldwide spread of antibiotic resistance is quickly becoming an increasingly concerning problem for human healthcare. Non-antibiotic antibacterial agents are in high demand for many Gram-negative bacterial pathogens, including Klebsiella pneumoniae. Klebsiella-targeting phages are among the most promising alternative therapy options. They have already been successfully applied in a number of cases, and it is expected that the need for anti-Klebsiella phages will only increase in the future. This prospect highlights the need for well-characterized therapeutic phages. In this work, we describe a K. pneumoniae phage, which also infects strains of Klebsiella oxytoca. Here, we characterize phage M198 in terms of its biological and genetic properties. Since in some phage therapy cases, phages are administered in combination with antibiotics, here, we also screen for possible synergistic effects of combining phage M198 with six different antibiotics. We found that phage M198 has good lytic activity against clinical isolates; it does not have any indications of a temperate lifestyle, and it has synergistic potential when combined with some therapeutically relevant antibiotics.

Antibiotic Resistance of Staphylococcus aureus Strains—Searching for New Antimicrobial Agents—Review

Inappropriate and excessive use of antibiotics is responsible for the rapid development of antimicrobial resistance, which is associated with increased patient morbidity and mortality. There is an urgent need to explore new antibiotics or alternative antimicrobial agents. S. aureus a commensal microorganism but is also responsible for numerous infections. In addition to innate resistance to β-lactam antibiotics, S. aureus strains resistant to methicillin (MRSA) often show resistance to other classes of antibiotics (multidrug resistance). The advancement of phage therapy against MRSA infections offers a promising alternative in the context of increasing antibiotic resistance. Therapeutic phages are easier to obtain and cheaper to produce than antibiotics. However, there is still a lack of standards to ensure the safe use of phages, including purification, dosage, means of administration, and the quantity of phages used. Some bacteria have developed defense mechanisms against phages. The use of phage cocktails or the combination of antibiotics and phages is preferred. For personalized therapy, it is essential to set up large collections to enable phage selection. In the future, the fight against MRSA strains using phages should be based on a multidisciplinary approach, including molecular biology and medicine. Other therapies in the fight against MRSA strains include the use of endolysin antimicrobial peptides (including defensins and cathelicidins). Researchers’ activities also focus on the potential use of plant extracts, honey, propolis, alkaloids, and essential oils. To date, no vaccine has been approved against S. aureus strains.

The pharmaceutical quality of freeze-dried tablets containing therapeutic bacteriophages against Pseudomonas aeruginosa and Staphylococcus aureus.

The preparation of a solid dosage form containing bacteriophages, which meets pharmaceutical requirements and ensures long-term stability of the phage effect, is significant for implementing phage therapy in practice. A commonly used method for processing phages into a solid form is freeze-drying into a (so-called) freeze-dried cake; however, to date there have been no studies examining the pharmacopeial parameters of freeze-dried tablets with bacteriophages. In this study, we describe the preparation and properties of freeze-dried tablets containing a cocktail of purified pseudomonal bacteriophage DSM 33593 from the genus Pbunavirus and staphylococcal bacteriophage DSM 33473 from the genus Kayvirus (108 PFU/tablet) as the active ingredient. Maltodextrin was used as a tablet filler, and D-mannitol was used as a cryoprotectant. The tablet preparation process resulted in a decrease in phage titer by no more than 1 log PFU/mL. For Pbunavirus, the titer values in tablet and liquid form were comparable. Kayvirus was more stable in tablet form than in liquid form after six months of storage at 25 °C (a decrease of 1.9 ± 0.8 log PFU/mL and 3.8 ± 0.7 log PFU/mL, respectively). The uniformity of mass of single-dose preparations, uniformity of content of single-dose preparations, and their disintegration complied with pharmacopeial requirements. The uniformity of dosage units of the tablets was maintained over three months. A microscopic examination of the internal part of the tablet revealed a heterogeneous structure, which does not affect the required pharmacopeial properties of the tablets. This study highlights the potential of freeze-dried tablets for long-term preservation of the phage effect at room temperature.

Bacteriophage induces modifications in outer membrane protein expression and antibiotic susceptibility in Acinetobacter baumannii.

Bacteriophages, the most abundant biological agents targeting bacteria, offer a promising alternative to antibiotics for combating multi-drug resistant pathogens like Acinetobacter baumannii. However, the rapid development of bacteriophage resistance poses a significant challenge. This study highlights the contribution of outer membrane proteins (OMPs) in the emergence of bacteriophage resistance in A. baumannii. The bacteriophage-sensitive and resistant isolates were studied for their native OMP profiles. Bacteriophage-tolerant A. baumannii were generated by infecting bacteria with bacteriophages and sub-culturing the survivors, and their expression of OMP and virulence was further characterized. These tolerant strains had significantly downregulated omp genes and under-expressed OMPs. Phenotypic changes like reduced adsorption to phages, deviant growth rates, biofilm-forming capacities, higher survival in limiting conditions, higher motility, and higher alkaline protease production were observed in the phage-tolerant strains equipped with better survival and virulent properties. The tolerant strains were re-sensitized to antibiotics they previously resisted. The significantly under-expressed OMPs in phage-tolerant strains were identified as OmpA and other OMPs similar to OmpA. This study could identify certain OMPs significantly under-expressed on bacteriophage exposure. The tolerant bacteria had altered phenotypic properties in addition to the development of phage resistance and the re-sensitisation to antibiotics, which paved the way for the future of phage therapeutics.

Phage vB_KlebPS_265 Active Against Resistant/MDR and Hypermucoid K2 Strains of Klebsiella pneumoniae

Klebsiella pneumoniae is an important opportunistic pathogen often resistant to antibiotics. Specific phages can be useful in eliminating infection caused by K. pneumoniae. Klebsiella phage vB_KlebPS_265 (KlebP_265) and its host strain were isolated from the sputum of a patient with Klebsiella infection. KlebP_265 was specific mainly to K. pneumoniae-type K2 strains including hypermucoid strains. Most of the hypermucoid KlebP_265-susceptible strains were antibiotic-resistant. This siphophage demonstrated good lytic activity and stability. The KlebP_265 genome was 46,962 bp and contained 88 putative genes; functions were predicted for 37 of them. No genes encoding integrases, toxins, or antibiotic resistance were found in the genome. So, KlebP_265 could potentially be a therapeutic phage. Comparative analysis indicated that KlebP_265 with the most relative Klebsiella phage DP01 formed the putative Dipiunovirus genus. Genome analysis revealed a large monophyletic group of phages related to KlebP_265 and DP01. This group is divided into two monophyletic clusters of phages forming new putative subfamilies Skatevirinae and Roufvirinae. Phylogenetic analysis showed extensive gene exchange between phages from the putative subfamilies. Horizontal transfer even involved conservative genes and led to clear genomic mosaicism, indicating multiple recombination events in the ancestral phages during evolution.

Translational research priorities for bacteriophage therapeutics.

The growing threat of antimicrobial resistant (AMR) bacterial pathogens coupled with the relative dearth of promising novel antibiotics requires the discovery and development additional medical interventions. Over the past decade bacteriophages have emerged one of the most promising new tools to combat AMR pathogens. Anecdotal clinical experiences under so-called 'compassionate use' regulatory pathways as well as a limited number of clinical trials have provided ample evidence of safety and early evidence of efficacy. For phages to reach their full potential it is critical that rigorous clinical trials be conducted that define their optimal use and that enable regulatory authorities to support the commercialization required to afford global access. The clinical development of phage therapeutics requires the design and execution of clinical trials that take full advantage of lessons learned from a century of antibiotic development and that use clinical investigation as a platform in which aspects of phage biology that are critical to therapeutics are more clearly elucidated. Translational research that elucidates phage biology in the context of clinical trials will promote highly relevant hypothesis-driven work in basic science laboratories and will greatly accelerate the development of the field of phage therapeutics.

Sxt1, Isolated from a Therapeutic Phage Cocktail, Is a Broader Host Range Relative of the Phage T3.

Using Escherichia coli BW25113 as a host, we isolated a novel lytic phage from the commercial poly-specific therapeutic phage cocktail Sextaphage® (Microgen, Russia). We provide genetic and phenotypic characterization of the phage and describe its host range on the ECOR collection of reference E. coli strains. The phage, hereafter named Sxt1, is a close relative of classical coliphage T3 and belongs to the Teetrevirus genus, yet its internal virion proteins, forming an ejectosome, differ from those of T3. In addition, the Sxt1 lateral tail fiber (LTF) protein clusters with those of the phages from the Berlinvirus genus. A comparison of T7, T3, and Sxt1 LTFs reveals the presence of insertions leading to the elongation of Sxt1 tail fibers, which, together with the difference in the HRDRs (host range-determining regions), might explain the expanded host specificity for the Sxt1.

The patentability of natural phages as therapeutics in the United States

The resurgence of phage therapy as a potent countermeasure against antimicrobial resistance has been accompanied by significant challenges in patent protection. This perspective paper examines the patentability of natural phages as therapeutics, focusing on the United States and extending to the European Union and Australia – jurisdictions at the forefront of this biotechnological innovation. We dissect the legal frameworks and identify the patent claims that have successfully navigated the complex intellectual property landscape. Our analysis reveals a dichotomy between the natural origin of therapeutic phages and the inventive steps required for patent eligibility. Despite hurdles, we highlight strategic innovations and specific patent claims that have been granted, suggesting a path forward for the commercialization and protection of phage therapy. We conclude with a call for a more adaptive legal framework to foster innovation and recognize the transformative potential of phage therapy in modern medicine.

Biological Characterization and Evaluation of the Therapeutic Value of Vibrio Phages 4141 and MJW Isolated from Clinical and Sewage Water Samples of Kolkata.

The growing prevalence of antimicrobial resistance (AMR) necessitates the development of new treatment methods to combat diseases like cholera. Lytic bacteriophages are viruses that specifically target and lyse bacteria upon infection, making them a possible treatment option for multi-drug-resistant pathogens. The current study investigated the potential role of bacteriophages isolated from clinical stool and sewage water samples in treating multi-drug-resistant Vibrio cholerae infection, finding that over 95% of the strains were susceptible. Whole-genome sequencing (WGS) analysis revealed that both Vibrio phage 4141 (4141) and Vibrio phage MJW (MJW) contain double-stranded DNA genomes consisting of 38,498 bp (43% GC) and 49,880 bp (42.5% GC) with 46 and 64 open reading frames (ORFs), respectively. Transmission electron microscope (TEM) and WGS analysis of Vibrio phage 4141 and Vibrio phage MJW validated that they are classified under the family Autographiviridae and Zobellviridae, respectively. Furthermore, both the phages showed highly significant biofilm degradation properties. The characterization of the phages and their strict host range, high spectrum of lytic ability, high efficiency of biofilm degradation, and close genetic similarity to the therapeutic phages indicates that these phages may be useful for therapeutic purposes for treating MDR V. cholerae infection in the future.

Trehalose polyphleates participate in Mycobacterium abscessus fitness and pathogenesis.

Mycobacteria produce a large repertoire of surface-exposed lipids with major biological functions. Among these lipids, trehalose polyphleates (TPPs) are instrumental in the infection of Mycobacterium abscessus by the therapeutic phage BPs. However, while the biosynthesis and transport of TPPs across the membrane by MmpL10 have been reported, the role of TPPs in host infection remains enigmatic. Here, we addressed whether the loss of TPPs influences interactions with macrophages and the virulence of M. abscessus. As anticipated, the deletion of mmpL10 in smooth (S) and rough (R) variants of M. abscessus abrogated TPP production, which was rescued upon gene complementation. Importantly, infection of human THP-1 cells with the mmpL10 mutants was associated with decreased intramacrophage survival and a reduced proportion of infected cells. The rough mmpL10 mutant showed an impaired capacity to block phagosomal acidification and was unable to co-localize with Galectin-3, a marker of phagosomal membrane damage. This suggests that TPPs participate, directly or indirectly, in phagolysosomal fusion and in phagosomal membrane damage to establish cytosolic communication. The TPP defect that affects the fitness and virulence of M. abscessus was further demonstrated in zebrafish embryos using a rough clinical strain resistant to phage BPs and harboring a frameshift mutation in mmpL10. Infection with this strain was correlated with a slight decrease in embryo survival and a reduced bacterial burden as compared to the corresponding parental and complemented derivatives. Together, these results indicate that TPPs are important surface lipids contributing to the pathogenicity of M. abscessus.IMPORTANCETrehalose polyphleates (TPPs) are complex lipids associated with the mycobacterial cell surface and were identified 50 years ago. While the TPP biosynthetic pathway has been described recently, the role of these lipids in the biology of mycobacteria remains yet to be established. The wide distribution of TPPs across mycobacterial species suggests that they may exhibit important functions in these actinobacteria. Here, we demonstrate that Mycobacterium abscessus, an emerging multidrug-resistant pathogen that causes severe lung diseases in cystic fibrosis patients, requires TPPs for survival in macrophages and virulence in a zebrafish model of infection. These findings support the importance of this underexplored family of lipids in mycobacterial pathogenesis.

Genomic surveillance as a scalable framework for precision phage therapy against antibiotic-resistant pathogens

Phage therapy is gaining increasing interest in the fight against critically antibiotic-resistant nosocomial pathogens. However, the narrow host range of bacteriophages hampers the development of broadly effective phage therapeutics and demands precision approaches. Here, we combine large-scale phylogeographic analysis with high-throughput phage typing to guide the development of precision phage cocktails targeting carbapenem-resistant Acinetobacter baumannii, a top-priority pathogen. Our analysis reveals that a few strain types dominate infections in each world region, with their geographical distribution remaining stable within 6 years. As we demonstrate in Eastern Europe, this spatiotemporal distribution enables preemptive preparation of region-specific phage collections that target most local infections. Finally, we showcase the efficacy of phage cocktails against prevalent strain types using invitro and animal infection models. Ultimately, genomic surveillance identifies patients benefiting from the same phages across geographical scales, thus providing a scalable framework for precision phage therapy.

Essential data for developing bacteriophage therapeutics

Essential data for developing bacteriophage therapeutics

Phage susceptibility testing methods or 'phagograms': where do we stand and where should we go?

Phage therapy is a highly promising approach to address the challenge that is presented by the global burden of antimicrobial resistance. Given the natural specificity of phages, phage susceptibility testing (PST) is a prerequisite for successful personalized therapy, allowing the selection of active phages from large and diverse collections. However, the issue of an easy-to-use and standardized technique remains. In this review, we describe the principles, advantages and drawbacks of two routinely used PST techniques: plaque and growth kinetic assays. These are labour-intensive and time-consuming methods that require automation of one or more steps, including preparation of test panels, incubation, reading and analysis of results. In addition to automation, there is an urgent need to establish a reference method to enable efficient of PST techniques selection of therapeutic phages. We discuss knowledge gaps and parameters that need to be investigated to work towards this goal.

Potential of training of anti-Staphylococcus aureus therapeutic phages against Staphylococcus epidermidis multidrug-resistant isolates is restricted by inter- and intra-sequence type specificity.

Phage therapy appears to be a promising approach to tackle multidrug-resistant bacteria, including staphylococci. However, most anti-staphylococcal phages have been characterized in Staphylococcus aureus, while a limited number of studies investigated phage activity against S. epidermidis. We studied the potential of phage training to extend the host range of two types of anti-S. aureus phages against S. epidermidis isolates. The Appelmans protocol was applied to a mixture of Kayvirus and a mixture of Silviavirus phages repeatedly exposed to seven S. epidermidis strains representative of nosocomial-associated sequence types (ST), including the world-wide disseminated ST2. We observed increased activity only for the Kayvirus mixture against two of these strains (ST2 or ST35). Phage subpopulations isolated from the training mixture using these two strains (five/strain) exhibited different evolved phenotypes, active only against their isolation strain or strains of the same ST. Of note, 16/47 ST2 strains were susceptible to one of the groups of trained phages. A comparative genomic analysis of ancestral and trained phage genomes, conducted to identify potential bacterial determinants of such specific activity, found numerous recombination events between two of the three ancestors. However, a small number of trained phage genes had nucleotide sequence modifications impacting the corresponding protein compared to ancestral phages, two to four of them per phage genome being specific of each group of phage subpopulations exhibiting different host range. The results suggest that anti-S. aureus phages can be adapted to S. epidermidis isolates but with inter- and intra-ST specificity.ImportanceS. epidermidis is increasingly recognized as a threat for public health. Its clinical importance is notably related to multidrug resistance. Phage therapy is one of the most promising alternative therapeutic strategies to antibiotics. Nonetheless, only very few phages active against this bacterial species have been described. In the present study, we showed that phage training can be used to extend the host range of polyvalent Kayvirus phages within the Staphylococcus genera to include S. epidermidis species. In the context of rapid development of phage therapy, in vitro forced adaptation of previously characterized phages could be an appealing alternative to fastidious repeated isolation of new phages to improve the therapeutic potential of a phage collection.

Multi-strain phage induced clearance of bacterial infections.

Bacteriophage (or 'phage' - viruses that infect and kill bacteria) are increasingly considered as a therapeutic alternative to treat antibiotic-resistant bacterial infections. However, bacteria can evolve resistance to phage, presenting a significant challenge to the near- and long-term success of phage therapeutics. Application of mixtures of multiple phage (i.e., 'cocktails') have been proposed to limit the emergence of phage-resistant bacterial mutants that could lead to therapeutic failure. Here, we combine theory and computational models of in vivo phage therapy to study the efficacy of a phage cocktail, composed of two complementary phages motivated by the example of Pseudomonas aeruginosa facing two phages that exploit different surface receptors, LUZ19v and PAK_P1. As confirmed in a Luria-Delbrück fluctuation test, this motivating example serves as a model for instances where bacteria are extremely unlikely to develop simultaneous resistance mutations against both phages. We then quantify therapeutic outcomes given single- or double-phage treatment models, as a function of phage traits and host immune strength. Building upon prior work showing monophage therapy efficacy in immunocompetent hosts, here we show that phage cocktails comprised of phage targeting independent bacterial receptors can improve treatment outcome in immunocompromised hosts and reduce the chance that pathogens simultaneously evolve resistance against phage combinations. The finding of phage cocktail efficacy is qualitatively robust to differences in virus-bacteria interactions and host immune dynamics. Altogether, the combined use of theory and computational analysis highlights the influence of viral life history traits and receptor complementarity when designing and deploying phage cocktails in immunocompetent and immunocompromised hosts.

Serratia marcescens ATCC 274 increases production of the red pigment prodigiosin in response to Chi phage infection

Serratia marcescens is an opportunistic human pathogen that produces a vibrant red pigment called prodigiosin. Prodigiosin has implications in virulence of S. marcescens and promising clinical applications. We discovered that addition of the virulent flagellotropic bacteriophage χ (Chi) to a culture of S. marcescens stimulates a greater than fivefold overproduction of prodigiosin. Active phage infection is required for the effect, as a χ-resistant strain lacking flagella does not respond to phage presence. Via a reporter fusion assay, we have determined that the addition of a χ-induced S. marcescens cell lysate to an uninfected culture causes a threefold increase in transcription of the pig operon, containing genes essential for pigment biosynthesis. Replacement of the pig promoter with a constitutive promoter abolished the pigmentation increase, indicating that regulatory elements present in the pig promoter likely mediate the phenomenon. We hypothesize that S. marcescens detects the threat of phage-mediated cell death and reacts by producing prodigiosin as a stress response. Our findings are of clinical significance for two main reasons: (i) elucidating complex phage-host interactions is crucial for development of therapeutic phage treatments, and (ii) overproduction of prodigiosin in response to phage could be exploited for its biosynthesis and use as a pharmaceutical.

Spatial propagation of temperate phages within and among biofilms.

Bacteria form groups comprised of cells and a secreted polymeric matrix that controls their spatial organization. These groups - termed biofilms - can act as refuges from environmental disturbances and from biotic threats, including phages. Despite the ubiquity of temperate phages and bacterial biofilms, live propagation of temperate phages within biofilms has never been characterized on cellular spatial scales. Here, we leverage several approaches to track temperate phages and distinguish between lytic and lysogenic host infections. We determine that lysogeny within E. coli biofilms initially occurs within a predictable region of cell group packing architecture on the biofilm periphery. Because lysogens are generally found on the periphery of large cell groups, where lytic viral infections also reduce local biofilm cell packing density, lysogens are predisposed to disperse into the passing liquid and are over-represented in biofilms formed from the dispersal pool of the original biofilm-phage system. Comparing our results with those for virulent phages reveals that temperate phages have previously unknown advantages in propagating over long spatial and time scales within and among bacterial biofilms.

High throughput platform technology for rapid target identification in personalized phage therapy

As bacteriophages continue to gain regulatory approval for personalized human therapy against antibiotic-resistant infections, there is a need for transformative technologies for rapid target identification through multiple, large, decentralized therapeutic phages biobanks. Here, we design a high throughput phage screening platform comprised of a portable library of individual shelf-stable, ready-to-use phages, in all-inclusive solid tablets. Each tablet encapsulates one phage along with luciferin and luciferase enzyme stabilized in a sugar matrix comprised of pullulan and trehalose capable of directly detecting phage-mediated adenosine triphosphate (ATP) release through ATP bioluminescence reaction upon bacterial cell burst. The tablet composition also enhances desiccation tolerance of all components, which should allow easier and cheaper international transportation of phages and as a result, increased accessibility to therapeutic phages. We demonstrate high throughput screening by identifying target phages for select multidrug-resistant clinical isolates of Pseudomonas aeruginosa, Salmonella enterica, Escherichia coli, and Staphylococcus aureus with targets identified within 30-120 min.

Isolation and Characterization of the Lytic Enterococcus faecium Phage φEc-ZZ2

Infections with vancomycin-resistant Enterococcus (VRE) have been increasingly reported, especially in immunocompromised patients. Bacteriophages (phages) are often considered as potential therapeutic candidates in treating infectious diseases. To search for therapeutic phages as antimicrobial agents for the treatment of Enterococcus faecium infections, eight E. faecium phages were isolated from the environments. One of these, phage φEc-ZZ2, which was capable of eliciting efficient lysis of E. faecium, was selected for analysis. Transmission electron microscopy revealed its resemblance to members of the family Siphoviridae, with a tail 200 nm long and an icosahedral head 45 nm in diameter. Phage φEc-ZZ2 exhibited rapid adsorption (> 99% adsorbed in 10 min), a short latency period of 20 min and a large burst size of 180 PFU/cell. Host range determination shows that φEc-ZZ2 has a relatively broader host range (specificity for 54.5% of tested E. faecium strains). Furthermore, when φEc-ZZ2 alone was incubated at different pH, the phage was stable over a wide pH range (5 to 9). These characteristics of φEc-ZZ2 will provide useful information for further research on the interaction between Enterococcus phages and their hosts. Moreover, in consideration of its pH stability and broader host range, φEc-ZZ2 may be a good candidate as a therapeutic/disinfectant agent to control nosocomial infections caused by E. faecium.

Study of Serratia phages using virulence tests

One of the main tests in selection of therapeutic phages is studing their virulence. Because of high stability of lysogenic status of bacterial culture, as well as ability in some cases of horizontal transfer of prophages and antibiotic resistance factors, existence of the moderate phages in medical preparations may cause negative results. The Serratia phages were studied with two main tests in the present work: sowing effectiveness and range of lysis action, as well as phage reproductive capacity on the host cells irradiated with UV rays. The results obtained showed that sowing effectiveness of studied phages and range of action are quite high. Out of 14 clones of Serratia phages 11 were characterized with reproductive ability on the host strains inactivated by UV rays. The results of above experimental works and other earlier tests allowed us selecting the components of Serratia phages for the medical-preventive preparation.