Preventive and therapeutic effects of a super-multivalent sialylated filamentous bacteriophage against the influenza virus.

The resurgence of influenza viruses as a significant global threat emphasizes the urgent need for innovative antiviral strategies beyond existing treatments. Here, we present the development and evaluation of a novel super-multivalent sialyllactosylated filamentous phage, termed t-6SLPhage, as a potent entry blocker for influenza A viruses. Structural variations in sialyllactosyl ligands, including linkage type, valency, net charge, and spacer length, were systematically explored to identify optimal binding characteristics against target hemagglutinins and influenza viruses. The selected SLPhage equipped with optimal ligands, exhibited exceptional inhibitory potency in in vitro infection inhibition assays. Furthermore, in vivo studies demonstrated its efficacy as both a preventive and therapeutic intervention, even when administered post-exposure at 2 days post-infection, under 4 lethal dose 50% conditions. Remarkably, co-administration with oseltamivir revealed a synergistic effect, suggesting potential combination therapies to enhance efficacy and mitigate resistance. Our findings highlight the efficacy and safety of sialylated filamentous bacteriophages as promising influenza inhibitors. Moreover, the versatility of M13 phages for surface modifications offers avenues for further engineering to enhance therapeutic and preventive performance.

Analysis of the life cycle of Helicobacter pylori bacteriophage KHP40 belonging to the genus Schmidvirus.

Bacteriophage (phage) KHP40 was previously isolated from the supernatant of a culture of Helicobacter pylori KMT83 cells. In this study, we analyzed the infection characteristics of KHP40, phage release pattern from KMT83 cells, and state of KHP40 DNA in KMT83 cells. The findings revealed that KHP40 phage showed varied adsorption efficiencies for different strains, long latent periods, and small burst sizes. Additionally, KHP40 activity was maintained at pH 2.5-12. KHP40 phages were released during the vegetative growth phase of the KMT83 cells. PCR analysis demonstrated that KHP40 DNA was stably maintained in KMT83 clones. Next-generation sequencing analysis revealed the presence of two distinct types of circular double-stranded DNA in H. pylori KMT83 cells. One was an H. pylori-specific DNA consisting of 1,578,403 bp, and the other was a 26,412 bp sequence that represented the episomal form of phage KHP40 DNA. Furthermore, defective KHP40-lysogenic DNA was detected in the H. pylori-specific DNA, the deleted portion of which appeared to have been transferred to another location in the bacterial genome. These findings indicate that KHP40 DNA exists in both episomal and defectively lysogenized states in KMT83 cells, and active phages are produced from KHP40-episomal DNA.

Evaluation of bacteriophages for the alleviation of potential bacterial contamination risks in developmental engineering.

This research aimed to address the potential bacterial contamination risks in developmental engineering (DE) using bacteriophages. To compare and contrast the exemplar Escherichia coli T4 and M13 bacteriophages, human dermal fibroblasts cultivated on culture plates, natural cellulosic scaffolds, and poly(methyl methacrylate) (PMMA) particles were utilized as two-dimensional (2D) cell, three-dimensional (3D) tissue, and modular tissue culture models, respectively. When directly introduced into these distinct culture systems, both phages survived, exhibited no significant effects on the cultured cells or tissues, yet displayed their potentials to alleviate the infections caused by corresponding bacterial host cells. Apart from direct addition into the culture medium, both phages were also coated on PMMA, polystyrene, poly(lactic acid) particles with different diameters (5, 10, 30, and 100 µm) and cellulosic scaffolds. The coated phages endured the coating processes and demonstrated their viabilities in plaque assays. Further testing indicated that the phages coated on the PMMA particles tolerated multiple deliberate rinses and centrifugations, but not thermal treatment at 60-80°C. In summary, T4 and M13 bacteriophages not only manifested their antibacterial functions in diverse 2D cell, 3D tissue, and modular tissue culture systems, but also demonstrated their potentials of coating modular scaffolds to alleviate the bacterial contamination risks in DE.

Characterization and Genome Analyses of the Novel Phages P2 and vB_AhydM-H1 Targeting Aeromonas hydrophila.

Background: The emergence of antibiotic-resistant Aeromonas hydrophila strains presents a global health and aquaculture challenge. Bacteriophages offer promise as an alternative to antibiotics for treating drug-resistant Aeromonas infections. Methods: Two new phages, P2 and vB_AhydM-H1, targeting pathogenic A. hydrophila were isolated from sewage water. Their morphology, growth characteristics, lytic activity, stability, and genomes were analyzed. Results: Phage P2, a member of genus Ahphunavirus, and vB_AhydM-H1, a novel member of genus Pahsextavirus, exhibited narrow host ranges, extended latent periods, and typical burst sizes. Both phages remained stable at 40°C for 1 h and within a pH range of 4 to 10 for 3 h. The genomes of P2 and vB_AhydM-H1 spanned 42,660 bp with 49 open reading frames (ORFs) and 52,614 bp with 72 ORFs, respectively. Proteomic (ViPTree) and phylogenetic (VICTOR) analyses confirmed that both phages aligned with their respective families. DeepTMHMM predictions suggested that P2 and vB_AhydM-H1 encode three and four ORFs with transmembrane domains, respectively. Conclusions: Safe for environmental and clinical use because of their lytic nature, and lack of virulence and resistance genes, these newly isolated phages expand the arsenal against antibiotic-resistant Aeromonas infections.

SalmoFree® Phage Additive Proves Its Safety for Laying Hens.

Background: The avian pathogen Salmonella Gallinarum causes avian typhosis in laying hens, leading to high mortality rates among adult birds, which poses a significant problem in the poultry industry. Various products, such as vaccines, antibiotics, probiotics, and disinfectants, are commonly used to prevent and control the disease on farms. An alternative to these products is the use of bacteriophages, which may effectively prevent the colonization of S. Gallinarum. Materials and Methods: This study evaluated the safety of SalmoFree®, a bacteriophage cocktail, administered to 276 laying hens from the first week of age until the 28th week. The hens were divided into two groups: a control group (138 birds) and a treatment group (138 birds). Over the 28-week period, eight doses of SalmoFree® (∼1010 UFP per bird) were administered via drinking water in a controlled environment. Results: The results indicate that the consumption of SalmoFree® has no adverse effects on bird health or zootechnical parameters. Additionally, there is a trend toward improving weight homogeneity (up to 19%), feed conversion (up to 68%), and egg weight (up to 2.7%). The detection of phages by PCR in cloacal swabs suggests that they persist in birds for 2 to 8 weeks post-ingestion. Furthermore, phages were detected in organs and eggshells, indicating that they provide protection beyond the gut. Conclusion: The study demonstrates that SalmoFree® is safe for use in laying hens and may offer additional benefits, such as improved zootechnical parameters and extended protection against S. Gallinarum through the persistence of bacteriophages in the birds.

Bacteriophages Against Bacterial Infections in Poultry Systems: A Scientometric Review.

Poultry production faces challenges from bacterial infections, aggravated by antibiotic resistance, affecting bird welfare and the industry's economy. Bacteriophages show promise as a solution, but their use in poultry systems is still limited. This study uses scientometric analysis to investigate the incidence of bacterial infections in poultry systems and bacteriophage application trends. The Web of Science database was used, and the articles were refined by searching for keywords that included the most rep orted bacteria in the different phases of poultry farming and the application of phages. The articles were analyzed using the CiteSpace and Excel software, allowing the evaluation of publication trends, influential countries, and correlations with antimicrobial resistance and the use of bacteriophages. Results highlight Escherichia coli prevalence in poultry systems and reveal a correlation between the number of publications and poultry productivity, with the United States and China leading both aspects. Findings offer insights into bacterial control gaps in poultry systems, underscoring the need for further research and practical strategies.

A Case of Persistent Intra-Abdominal Stenotrophomonas maltophilia Infection Despite Bacteriophage Therapy.

Multidrug resistant infections are a challenge in the health care setting and a cause of patient morbidity and mortality. Bacteriophages (phages) are viruses that target and kill bacteria and have been used in patients to treat bacterial infections. We present a case of disseminated Stenotrophomonas maltophilia infection, with pulmonary, intra-abdominal and bloodstream involvement. The patient was treated with a combination of antibiotics and personalized phage therapy, administered daily for 12 days both intravenously as well as via intra-abdominal drains. Phage therapy was well-tolerated, the patient cleared S. maltophilia from their bloodstream and their intra-abdominal abscesses were stable or decreased in size. However, the intra-abdominal fluid cultures remained positive for S. maltophilia. Unfortunately, the patient passed away 2 months after completion of phage therapy due to multiorgan failure. These data highlight the difficulty of treating critically ill patients and clearing complex, biofilm mediated infections, even with phages. More information is needed regarding the optimal treatment protocols for phage therapy in complex multifocal infections.

A Vibrio cholerae anti-phage system depletes nicotinamide adenine dinucleotide to restrict virulent bacteriophages.

Bacteria and their predatory viruses (bacteriophages or phages) are in a perpetual molecular arms race. This has led to the evolution of numerous phage defensive systems in bacteria that are still being discovered, as well as numerous ways of interference or circumvention on the part of phages. Here, we identify a unique molecular battle between the classical biotype of Vibrio cholerae and virulent phages ICP1, ICP2, and ICP3. We show that classical biotype strains resist almost all isolates of these phages due to a 25-kb genomic island harboring several putative anti-phage systems. We observed that one of these systems, Nezha, encoding SIR2-like and helicase proteins, inhibited the replication of all three phages. Bacterial SIR2-like enzymes degrade the essential metabolic coenzyme nicotinamide adenine dinucleotide (NAD+), thereby preventing replication of the invading phage. In support of this mechanism, we identified one phage isolate, ICP1_2001, which circumvents Nezha by encoding two putative NAD+ regeneration enzymes. By restoring the NAD+ pool, we hypothesize that this system antagonizes Nezha without directly interacting with its proteins and should be able to antagonize other anti-phage systems that deplete NAD+.IMPORTANCEBacteria and phages are in a perpetual molecular arms race, with bacteria evolving an extensive arsenal of anti-phage systems and phages evolving mechanisms to overcome these systems. This study identifies a previously uncharacterized facet of the arms race between Vibrio cholerae and its phages. We identify an NAD+-depleting anti-phage defensive system called Nezha, potent against three virulent phages. Remarkably, one phage encodes proteins that regenerate NAD+ to counter the effects of Nezha. Without Nezha, the NAD+ regeneration genes are detrimental to the phage. Our study provides new insight into the co-evolutionary dynamics between bacteria and phages and informs the microbial ecology and phage therapy fields.

Complete genome sequences of three Pseudomonas aeruginosa jumbo bacteriophages discovered in Kenya.

The genomes of three Pseudomonas aeruginosa Phikzvirus bacteriophages isolated in Kenya are described. The genomes of phages vB_PaePAO1-KEN19, vB_Pae3705-KEN49, and vB_Pae10145-KEN51, respectively, had lengths of 278,921, 280,231, and 280,173 bp, with 36.93%, 36.84%, and 36.86% GC content, containing 419, 417, and 417 coding sequences (including seven tRNAs in each genome).

Complete genome sequence of Rhodococcus qingshengii phage Perlina.

Rhodococcus phage Perlina is a novel phage isolated on Rhodococcus qingshengii S10. Perlina encodes 112 open reading frames with typical phage structural genes identified and 3 tRNAs (tRNA-Ile, tRNA-Met, and tRNA-Asn). Few close relatives can be identified at the nucleotide level, suggesting a new phage species.

Long-Term Effectiveness of Engineered T7 Phages Armed with Silver Nanoparticles Against Escherichia coli Biofilm.

The escalating threat of antibiotic-resistant bacteria, particularly those forming biofilm structures, underscores the urgent need for alternative treatment strategies. Bacteriophages have emerged as promising agents for combating bacterial infections, especially those associated with biofilm formation. However, the efficacy of phage therapy can be limited by the development of bacterial resistance and biofilm regrowth. Interestingly, phages could be combined with other agents, such as metal nanoparticles, to enhance their antibacterial effectiveness. Since the therapeutic strategy of using phages and metal nanoparticles has been developed relatively recently, evaluating its efficacy under various conditions is essential, with a particular focus on the duration of activity. This study tested the hypothesis that a novel approach to combating bacterial biofilms, based on phages armed with silver nanoparticles (AgNPs), would exhibit enhanced activity over an extended period after application. In this work, we investigated the potential of engineered T7 phages armed with AgNPs for eradicating Escherichia coli biofilm. We demonstrated that such biomaterial exhibits sustained antimicrobial activity even after prolonged exposure. Compared to phages alone or AgNPs alone, the biomaterial significantly enhances biofilm eradication, particularly after 48 hours of treatment. These findings highlight the potential of synergistic phage-nanoparticle strategies for combatting biofilm-associated infections.

Klebsiella pneumoniae infections and phage therapy.

OBJECTIVE: Carbapenem-colistin-resistant Klebsiella pneumoniae has emerged as a serious global problem. Klebsiella pneumoniae is a major culprit in healthcare settings and is responsible for septicemia, urinary tract infections, pneumonia, meningitis, burn wound and surgical site infections, and liver abscesses even in younger and healthier population worldwide. The formation of biofilm prevents antibiotics from reaching the bacteria and exerting their effector mechanism. The non-availability of therapeutic alternatives (antibiotic therapy) further complicates the scenario. However, in the era of antibiotic resistance, bacteriophage therapy emerges as a ray of hope against antibiotic-resistant bacteria. METHOD: The present review focuses on the therapeutic potential of bacteriophages as an antimicrobial agent with special reference to safety, specificity, efficacy, dosage, and dosage frequency against Pan-Drug Resistant (PDR) K. pneumoniae, both in-vitro and in-vivo (animals and human) studies. RESULT: This review highlights the perspectives therapeutic potential of bacteriophages, their impact on the host immune system, combination therapy, and bacteriophage-encoded gene product endolysin, artificial lysins (Artilysins), polysaccharide depolymerase, and peptidoglycan hydrolases. CONCLUSION: This review briefly describes the application of bacteriophage and its encoded gene products in clinical trials.

Rapid design of bacteriophage cocktails to suppress the burden and virulence of gut-resident carbapenem-resistant Klebsiella pneumoniae.

Antibiotic use can lead to the expansion of multi-drug-resistant pathobionts within the gut microbiome that can cause life-threatening infections. Selective alternatives to conventional antibiotics are in dire need. Here, we describe a Klebsiella PhageBank for the tailored design of bacteriophage cocktails to treat multi-drug-resistant Klebsiella pneumoniae. Using a transposon library in carbapenem-resistant K. pneumoniae, we identify host factors required for phage infection in major Klebsiella phage families. Leveraging the diversity of the PhageBank, we formulate phage combinations that eliminate K. pneumoniae with minimal phage resistance. Optimized cocktails selectively suppress the burden of K. pneumoniae in the mouse gut and drive the loss of key virulence factors that act as phage receptors. Phage-mediated diversification of bacterial populations in the gut leads to co-evolution of phage variants with higher virulence and broader host range. Altogether, the Klebsiella PhageBank charts a roadmap for phage therapy against a critical multidrug-resistant human pathogen.

Truncated M13 phage for smart detection of E. coli under dark field.

BACKGROUND: The urgent need for affordable and rapid detection methodologies for foodborne pathogens, particularly Escherichia coli (E. coli), highlights the importance of developing efficient and widely accessible diagnostic systems. Dark field microscopy, although effective, requires specific isolation of the target bacteria which can be hindered by the high cost of producing specialized antibodies. Alternatively, M13 bacteriophage, which naturally targets E. coli, offers a cost-efficient option with well-established techniques for its display and modification. Nevertheless, its filamentous structure with a large length-diameter ratio contributes to nonspecific binding and low separation efficiency, posing significant challenges. Consequently, refining M13 phage methodologies and their integration with advanced microscopy techniques stands as a critical pathway to improve detection specificity and efficiency in food safety diagnostics. METHODS: We employed a dual-plasmid strategy to generate a truncated M13 phage (tM13). This engineered tM13 incorporates two key genetic modifications: a partial mutation at the N-terminus of pIII and biotinylation at the hydrophobic end of pVIII. These alterations enable efficient attachment of tM13 to diverse E. coli strains, facilitating rapid magnetic separation. For detection, we additionally implemented a convolutional neural network (CNN)-based algorithm for precise identification and quantification of bacterial cells using dark field microscopy. RESULTS: The results obtained from spike-in and clinical sample analyses demonstrated the accuracy, high sensitivity (with a detection limit of 10 CFU/µL), and time-saving nature (30 min) of our tM13-based immunomagnetic enrichment approach combined with AI-enabled analytics, thereby supporting its potential to facilitate the identification of diverse E. coli strains in complex samples. CONCLUSION: The study established a rapid and accurate detection strategy for E. coli utilizing truncated M13 phages as capture probes, along with a dark field microscopy detection platform that integrates an image processing model and convolutional neural network.

Bacteriophage P2-71: a promising therapeutic against multidrug-resistant Proteus mirabilis in urinary tract infections.

Background: Proteus mirabilis is a Gram-negative, rod-shaped bacterium widely found in natural environments. It is known for causing a range of severe illnesses in mammals, particularly urinary tract infections (UTIs). This study evaluates the therapeutic efficacy of phage P2-71 against Proteus mirabilis in vivo and in vitro environments. Methods: The in vitro therapeutic potential of bacteriophage P2-71 was assessed through the ability of phage to kill Proteus mirabilis by using a plate counting assay, and biofilm inhibition and biofilm lysis assays using a microtitre plate method. Additionally, an in vivo UTI model in C57BL/6Jmice was developed via urethral inoculation of the bacterium. Phage therapy was administered through urethral injection over a period of 5 days. Therapeutic outcomes were measured by analyzing bacterial load, phage titer, inflammatory markers, and histopathological changes in the urine, urogenital tissues, and spleen. Results: In vitro, bacteriophage P2-71 achieved significant reductions in P. mirabilis concentrations, with log reductions of 1.537 and 0.7009 CFU/mL in laboratory and urine environments, respectively (p < 0.001). The phage also decreased biofilm formation by 34-49% and lysed 15-25% of mature biofilms at various multiplicities of infection (MOIs) (p < 0.001). In vivo, phage treatment significantly lowered bacterial concentrations in the urine on Days 1 and 3 (p < 0.0001), achieving a maximum reduction of 4.602 log10 CFU/mL; however, its effectiveness diminished by Day 5 (p > 0.05). Concurrently, phage titers decreased over time. Importantly, phage treatment notably reduced bacterial load in the bladder, kidneys, and spleen (p < 0.001). Inflammatory markers such as IL-6, IL-1ß, and TNF-α were significantly lower in the treatment group, especially in the bladder (p < 0.0001), indicating an effective reduction in inflammation. Histopathological analysis showed significant mitigation of tissue damage. Conclusion: The results demonstrated that bacteriophage P2-71 is a promising alternative therapy for UTIs caused by MDR Proteus mirabilis. This bacteriophage therapy offers a viable strategy for managing infections where traditional antimicrobials fail, highlighting its potential in clinical applications.

Ejectosome of Pectobacterium bacteriophage ΦM1.

Podophages that infect gram-negative bacteria, such as Pectobacterium pathogen ΦM1, encode tail assemblies too short to extend across the complex gram-negative cell wall. To overcome this, podophages encode a large protein complex (ejectosome) packaged inside the viral capsid and correspondingly ejected during infection to form a transient channel that spans the periplasmic space. Here, we describe the ejectosome of bacteriophage ΦM1 to a resolution of 3.32 Å by single-particle cryo-electron microscopy (cryo-EM). The core consists of tetrameric and octameric ejection proteins which form a ∼1.5-MDa ejectosome that must transition through the ∼30 Å aperture created by the short tail nozzle assembly that acts as the conduit for the passage of DNA during infection. The ejectosome forms several grooves into which coils of genomic DNA are fit before the DNA sharply turns and goes down the tunnel and into the portal. In addition, we reconstructed the icosahedral capsid and hybrid tail apparatus to resolutions between 3.04 and 3.23 Å, and note an uncommon fold adopted by the dimerized decoration proteins which further emphasize the structural diversity of podophages. These reconstructions have allowed the generation of a complete atomic model of the ΦM1, uncovering two distinct decoration proteins and highlighting the exquisite structural diversity of tailed bacteriophages.

Phage therapy: A novel approach against multidrug-resistant pathogens.

The rapid rise of multidrug-resistant (MDR) organisms has created a critical need for alternative treatment options. Phage therapy is gaining attention as an effective way to fight bacterial infections by using lytic bacteriophages to specifically target and kill harmful bacteria. This review discusses several phage therapeutic options and emphasizes new developments in phage biology. Phage treatment has proven to be successful against MDR bacteria, as evidenced by multiple human clinical trials that indicate favorable results in treating a range of diseases caused by these pathogens. Despite these promising results, challenges such as phage resistance, regulatory hurdles, and the need for standardized treatment protocols remain. To effectively combat MDR bacterial infections, future research must focus on enhancing phage effectiveness, guaranteeing safety for human usage and incorporating phage therapy into clinical practice.

Case report: Personalized triple phage-antibiotic combination therapy to rescue necrotizing fasciitis caused by Panton-Valentine leukocidin-producing MRSA in a 12-year-old boy.

Maximal standard-of-care (SOC) management could not stop the life-threatening progression of a necrotizing fasciitis induced by Panton-Valentine Leukocidin-producing Methicillin-Resistant Staphylococcus aureus (MRSA) in a 12-year-old boy. Multi-route phage therapy was initiated along with antibiotics against Staphylococcus aureus, Pseudomonas aeruginosa and Stenotrophomonas maltophilia, eventually leading to full recovery with no reported adverse events.

Recruitment of complete crAss-like phage genomes reveals their presence in chicken viromes, few human-specific phages, and lack of universal detection.

The order Crassvirales, which includes the prototypical crAssphage (p-crAssphage), is predominantly associated with humans, rendering it the most abundant and widely distributed group of DNA phages in the human gut. The reported human specificity and wide global distribution of p-crAssphage makes it a promising human fecal marker. However, the specificity for the human gut as well as the geographical distribution around the globe of other members of the order Crassvirales remains unknown. To determine this, a recruitment analysis using 91 complete, non-redundant genomes of crAss-like phages in human and animal viromes revealed that only 13 crAss-like phages among the 91 phages analyzed were highly specific to humans, and p-crAssphage was not in this group. Investigations to elucidate whether any characteristic of the phages was responsible for their prevalence in humans showed that the 13 human crAss-like phages do not share a core genome. Phylogenomic analysis placed them in three independent families, indicating that within the Crassvirales group, human specificity is likely not a feature of a common ancestor but rather was introduced on separate/independent occasions in their evolutionary history. The 13 human crAss-like phages showed variable geographical distribution across human metagenomes worldwide, with some being more prevalent in certain countries than in others, but none being universally identified. The varied geographical distribution and the absence of a phylogenetic relationship among the human crAss-like phages are attributed to the emergence and dissemination of their bacterial host, the symbiotic human strains of Bacteroides, across various human populations occupying diverse ecological niches worldwide.

Overcoming Bacteriophage Contamination in Bioprocessing: Strategies and Applications.

Bacteriophage contamination has a devastating impact on the viability of bacterial hosts and can significantly reduce the productivity of bioprocesses in biotechnological industries. The consequences range from widespread fermentation failure to substantial economic losses, highlighting the urgent need for effective countermeasures. Conventional prevention methods, which focus primarily on the physical removal of bacteriophages from equipment, bioprocess units, and the environment, have proven ineffective in preventing phage entry and contamination. The coevolutionary dynamics between phages and their bacterial hosts have spurred the development of a diverse repertoire of antiviral defense mechanisms within microbial communities. These naturally occurring defense strategies can be harnessed through genetic engineering to convert phage-sensitive hosts into robust, phage-resistant cell factories, providing a strategic approach to mitigate the threats posed by bacteriophages to industrial bacterial processes. In this review, an overview of the various defense strategies and immune systems that curb the propagation of bacteriophages and highlight their applications in fermentation bioprocesses to combat phage contamination is provided. Additionally, the tactics employed by phages to circumvent these defense strategies are also discussed, as preventing the emergence of phage escape mutants is a key component of effective contamination management.