Application Of Bacteriophages Research Articles

The Safety of Bacteriophages in Treatment of Diseases Caused by Multidrug-Resistant Bacteria.

Given the urgency due to the rapid emergence of multidrug-resistant (MDR) bacteria, bacteriophages (phages), which are viruses that specifically target and kill bacteria, are rising as a potential alternative to antibiotics. In recent years, researchers have begun to elucidate the safety aspects of phage therapy with the aim of ensuring safe and effective clinical applications. While phage therapy has generally been demonstrated to be safe and tolerable among animals and humans, the current research on phage safety monitoring lacks sufficient and consistent data. This emphasizes the critical need for a standardized phage safety assessment to ensure a more reliable evaluation of its safety profile. Therefore, this review aims to bridge the knowledge gap concerning phage safety for treating MDR bacterial infections by covering various aspects involving phage applications, including phage preparation, administration, and the implications for human health and the environment.

Bacteriophages with depolymerase activity in the control of antibiotic resistant Klebsiella pneumoniae biofilms

Klebsiella pneumoniae is associated with a variety of infections, such as pneumonia, urogenital infection, liver abscess, and bloodstream infection. It is especially dangerous for patients in medical facilities, where it can cause ventilator-associated pneumonia or intensive care unit-acquired pneumonia. The emergence of multidrug-resistant and hypervirulent strains as well as the ability to form biofilms on various medical devices complicates the treatment of such infections and makes the use of antibiotics ineffective. The application of bacteriophages is a promising alternative for combating Klebsiella pneumoniae biofilms. In the present study a cocktail of 3 bacteriophages with depolymerase activity was used to control antibiotic resistant Klebsiella pneumoniae biofilms in vitro. Biofilms were examined using optical and scanning electron microscopy. The obtained results demonstrate that the studied bacteriophage cocktail can effectively disrupt Klebsiella pneumoniae biofilms.

The Biotechnological Application of Bacteriophages: What to Do and Where to Go in the Middle of the Post-Antibiotic Era.

Amid the escalating challenges of antibiotic resistance, bacterial infections have emerged as a global threat. Bacteriophages (phages), viral entities capable of selectively infecting bacteria, are gaining momentum as promising alternatives to traditional antibiotics. Their distinctive attributes, including host specificity, inherent self-amplification, and potential synergy with antibiotics, render them compelling candidates. Phage engineering, a burgeoning discipline, involves the strategic modification of bacteriophages to enhance their therapeutic potential and broaden their applications. The integration of CRISPR-Cas systems facilitates precise genetic modifications, enabling phages to serve as carriers of functional genes/proteins, thereby enhancing diagnostics, drug delivery, and therapy. Phage engineering holds promise in transforming precision medicine, addressing antibiotic resistance, and advancing diverse applications. Emphasizing the profound therapeutic potential of phages, this review underscores their pivotal role in combatting bacterial diseases and highlights their significance in the post-antibiotic era.

A Bacteriophage Cocktail Reduces Five Relevant Salmonella Serotypes at Low Multiplicities of Infection and Low Temperatures.

Salmonella are important pathogenic bacteria and, following Campylobacter, they are the second most common cause of bacterial foodborne infections worldwide. To reduce the presence of bacteria along the food chain, the application of bacteriophages (phages) may be a promising tool. In this study, the lytic properties of six phages against five relevant Salmonella serotypes (S. Enteritidis, S. Typhimurium, S. Infantis, S. Paratyphi B and S. Indiana) were analyzed. Three phages were able to lyse all five serotypes. We determined the lytic potential of each phage on indicator strains in vitro at room temperature (RT) and at 37 °C using low multiplicities of infection (MOIs). Most phages reduced their host more efficiently at RT than at 37 °C, even at the lowest MOI of 0.001. Following this, the lytic activity of a cocktail comprising five phages (MOI = 0.1) was examined with each of the five serotypes and a mix of them at RT, 15, 12, 10, 8 and 6 °C. All cultures of single serotypes as well as the mixture of strains were significantly reduced at temperatures as low as 8 °C. For single serotypes, reductions of up to 5 log10 units and up to 2.3 log10 units were determined after 6 h (RT) and 40 h (8 °C), respectively. The mixture of strains was reduced by 1.7 log10 units at 8 °C. The data clearly suggest that these phages are suitable candidates for biocontrol of various Salmonella serotypes under food manufacturing conditions.

Prevention and Control of Human Salmonella enterica Infections: An Implication in Food Safety.

Salmonella is a foodborne zoonotic pathogen causing diarrhoeal disease to humans after consuming contaminated water, animal, and plant products. The bacterium is the third leading cause of human death among diarrhoeal diseases worldwide. Therefore, human salmonellosis is of public health concern demanding integrated interventions against the causative agent, Salmonella enterica. The prevention of salmonellosis in humans is intricate due to several factors, including an immune-stable individual infected with S. enterica continuing to shed live bacteria without showing any clinical signs. Similarly, the asymptomatic Salmonella animals are the source of salmonellosis in humans after consuming contaminated food products. Furthermore, the contaminated products of plant and animal origin are a menace in food industries due to Salmonella biofilms, which enhance colonization, persistence, and survival of bacteria on equipment. The contaminated food products resulting from bacteria on equipment offset the economic competition of food industries and partner institutions in international business. The most worldwide prevalent broad-range Salmonella serovars affecting humans are Salmonella Typhimurium and Salmonella Enteritidis, and poultry products, among others, are the primary source of infection. The broader range of Salmonella serovars creates concern over multiple strategies for preventing and controlling Salmonella contamination in foods to enhance food safety for humans. Among the strategies for preventing and controlling Salmonella spread in animal and plant products include biosecurity measures, isolation and quarantine, epidemiological surveillance, farming systems, herbs and spices, and vaccination. Other measures are the application of phages, probiotics, prebiotics, and nanoparticles reduced and capped with antimicrobial agents. Therefore, Salmonella-free products, such as beef, pork, poultry meat, eggs, milk, and plant foods, such as vegetables and fruits, will prevent humans from Salmonella infection. This review explains Salmonella infection in humans caused by consuming contaminated foods and the interventions against Salmonella contamination in foods to enhance food safety and quality for humans.

Optimization of the Use of a Commercial Phage-Based Product as a Control Strategy of Listeria monocytogenes in the Fresh-Cut Industry.

A commercial phage biocontrol for reducing Listeria monocytogenes has been described as an effective tool for improving fresh produce safety. Critical challenges in the phage application must be overcome for the industrial application. The validation studies were performed in two processing lines of two industry collaborators in Spain and Denmark, using shredded iceberg lettuce as the ready-to-eat (RTE), high process volume product. The biocontrol treatment optimized in lab-scale trials for the application of PhageGuard ListexTM was confirmed in industrial settings by four tests, two in Spain and two in Denmark. Results showed that the method of application that included the device and the processing operation step was appropriate for the proper application. The proper dose of Phage Guard ListexTM was reached in shredded iceberg lettuce and the surface was adequately covered for the successful application of phages. There was no impact on the headspace gas composition (CO2 and O2 levels), nor on the color when untreated and treated samples were compared. The post-process treatment with PhageGuard ListexTM did not cause any detrimental impact on the sensory quality, including flavor, texture, browning, spoilage, and visual appearance over the shelf-life as the phage solution was applied as a fine, mist solution.

RESULTS OF THE USE OF POLYVALENT BACTERIOPHAGES IN THE TREATMENT OF INFECTIOUS COMPLICATIONS IN EMERGENCY ABDOMINAL SURGERY

We comparatively studied the results of treatment of 86 patients with surgical infectious complications after cavitary intra-abdominal operations for emergency abdominal diseases. In the main group, treatment was supplemented with polyvalent bacteriophages. We studied integral scales for assessing the severity of patient’s condition of (APACHE II, ACI, ASEPSIS), microbial pattern, enteral morphofunctional coefficient, levels of alkaline phosphatase and intestinal alkaline phosphatase, and indicators of cellular immunity. The results of the study show that complex treatment including polyvalent bacteriophages in postoperative purulent-inflammatory complications in urgent surgery reduces the development of antibiotic-resistant strains, as well as the risk of nosocomial microbial wound associations. Enteral application of polyvalent bacteriophages in patients with acute intestinal failure syndrome normalizes microbial enteral imbalance and the level of pathogens contamination of the small intestine. Supplementing the treatment of postoperative abdominal purulent-inflammatory complications with phage therapy can reduce the number of surgical interventions, as well as the treatment duration and mortality.

Bacteriophages targeting protective commensals impair resistance against Salmonella Typhimurium infection in gnotobiotic mice.

Gut microbial communities protect the host against a variety of major human gastrointestinal pathogens. Bacteriophages (phages) are ubiquitous in nature and frequently ingested via food and drinking water. Moreover, they are an attractive tool for microbiome engineering due to the lack of known serious adverse effects on the host. However, the functional role of phages within the gastrointestinal microbiome remain poorly understood. Here, we investigated the effects of microbiota-directed phages on infection with the human enteric pathogen Salmonella enterica serovar Typhimurium (S. Tm), using a gnotobiotic mouse model (OMM14) for colonization resistance (CR). We show, that phage cocktails targeting Escherichia coli and Enterococcus faecalis acted in a strain-specific manner. They transiently reduced the population density of their respective target before establishing coexistence for up to 9 days. Infection susceptibility to S. Tm was markedly increased at an early time point after challenge with both phage cocktails. Surprisingly, OMM14 mice were also susceptible 7 days after a single phage inoculation, when the targeted bacterial populations were back to pre-phage administration density. Concluding, our work shows that phages that dynamically modulate the density of protective members of the gut microbiota can provide opportunities for invasion of bacterial pathogens, in particular at early time points after phage application. This suggests, that phages targeting protective members of the microbiota may increase the risk for Salmonella infection.

Prospects for the application of bacteriophages in the treatment of sinusitis caused by antibiotic-resistant bacterial strains

Purulent diseases in otorhinolaryngology are of great interest due to their widespread prevalence and the objective difficulties of treatment. One of the most frequently encountered nosological forms is purulent sinusitis. A key element in the complex treatment of bacterial sinusitis is systemic antibiotic therapy aimed at the eradication of pathobionts from the focus of infection. However, the increasing worldwide drug resistance of etiologically significant microorganisms complicates antibacterial chemotherapy of purulent sinusitis and raises questions about its future feasibility. The threat of a change from a resistance era to a post-antibiotic era is very significant, given the acute shortage of new antibiotics and the relatively rapid adaptation of bacterial microorganisms to them. The global community is therefore faced with the challenge of finding other drugs with antimicrobial activity. In recent decades, genetically modified virulent bacteriophages and phagolytes isolated on their basis are becoming increasingly attractive in the fight against purulent infections of various localizations caused by drug-resistant bacteria. This literature review focuses on the potential of phage therapy for acute bacterial sinusitis in an era of globally increasing bacterial strain resistance. The article details the causative agents of community-acquired and hospital-acquired acute sinusitis, the main mechanisms of bacterial antibiotic resistance, the effect of virulent phages on bacteria, and studies on the efficacy of phage therapy in the treatment of acute sinusitis. The facts outlined herein clearly indicate the efficacy of phage therapy, as well as therapeutic perspectives on using bacteriophages in the complex treatment of purulent sinusitis caused by antibiotic-resistant microbiota.

Sequential treatment effects on phage–antibiotic synergistic application against multi-drug-resistant Acinetobacter baumannii

Bacteriophage (phage) therapy, exploiting phages which are the natural enemies of bacteria, has been re-introduced to treat multidrug-resistant (MDR) bacterial infections. However, some intrinsic drawbacks of phages are overshadowing their clinical use, particularly the narrow host spectrum and rapid emergence of resistance upon treatment. The use of phage-antibiotic combinations exhibiting synergistic bacterial killing [termed 'phage-antibiotic synergy' (PAS)] has therefore been proposed. It is well reported that the types and doses of phages and antibiotics are critical in achieving PAS. However, the impact of treatment order has received less research attention. As such, this study used an Acinetobacter baumannii phage vB_AbaM-IME-AB2 and colistin as a model PAS combination to elucidate the order effects in-vitro. While application of the phage 8 h before colistin treatment demonstrated the greatest antibacterial synergy, it failed to prevent the development of phage resistance. On the other hand, simultaneous application and antibiotic followed by phage application were able to suppress/delay the development of resistance effectively, and simultaneous application demonstrated superior antibacterial and antibiofilm activities. Further in-vivo investigation is required to confirm the impact of treatment order on PAS.

Isolation, characterization, and evaluation of putative new bacteriophages for controlling bacterial spot on tomato in Brazil.

Bacterial spot is a highly damaging tomato disease caused by members of several species of the genus Xanthomonas. Bacteriophages have been studied for their potential use in the biological control of bacterial diseases. In the current study, bacteriophages were obtained from soil and tomato leaves in commercial fields in Brazil with the aim of obtaining biological control agents against bacterial spot. Phage isolation was carried out by co-cultivation with isolates of Xanthomonas euvesicatoria pv. perforans, which was prevalent in the collection areas. In a host range evaluation, none of the phage isolates was able to induce a lytic cycle in all of the bacterial isolates tested. In in vivo tests, treatment of susceptible bacterial isolates with the corresponding phage prior to application to tomato plants led to a reduction in the severity of the resulting disease. The level of disease control provided by phage application was equal to or greater than that achieved using copper hydroxide. Electron microscopy analysis showed that all of the phages had similar morphology, with head and tail structures similar to those of viruses belonging to the class Caudoviricetes. The presence of short, non-contractile tubular tails strongly suggested that these phages belong to the family Autographiviridae. This was confirmed by phylogenetic analysis, which further revealed that they all belong to the genus Pradovirus. The phages described here are closely related to each other and potentially belong to a new species within the genus. These phages will be evaluated in future studies against other tomato xanthomonad strains to assess their potential as biological control agents.

Bacteriophage cocktail application for Campylobacter mitigation - from in vitro to in vivo

BackgroundEffective strategies are urgently needed to control Campylobacteriosis, one of the most important foodborne gastrointestinal diseases worldwide. Administering bacteriophages (phages) is under evaluation as a possible intervention strategy in primary poultry production to reduce the public health risk of human infection. A major challenge is the translation of results from small-scale animal studies to large broiler flocks. In this study, the in vitro lytic activity of 18 Campylobacter-specific group II phages and 19 group III phages were examined singly, and in different combinations from the same group and from both groups using a planktonic killing assay. Based on these results, a combination of phage NCTC 12,673 (group III) and vB_CcM-LmqsCPL1/1 (group II) was selected for in vivo application in a seeder bird model to study its effectiveness under conditions as close as possible to field conditions. One hundred eighty Ross 308 broiler chickens were divided into a control and a treatment group. Ten days post hatch, seeder birds were orally inoculated with the C. jejuni target strain. Phages were administered via drinking water at a total concentration of 107 PFU/mL four, three, and two days before necropsy.ResultsCombining group II and group III phages resulted in significantly higher in vitro growth inhibition against the C. jejuni target strain BfR-CA-14,430 than single application or combinations of phages from the same group. The results of the animal trial showed that the application of the two phages significantly reduced Campylobacter counts in cloacal swabs. At necropsy, Campylobacter counts in colonic content of the treatment group were significantly reduced by 2 log10 units compared to the control group.ConclusionsWe demonstrated that combining phages of groups II and III results in significantly increased lytic activities. The in vitro results were successfully translated into practical application in a study design close to field conditions, providing new data to apply phages in conventional broiler flocks in the future. Phage application reduced the fecal Campylobacter excretion and Campylobacter concentrations in the colon of broilers.

Applications of Bacteriophage and Its Current Status

Applications of Bacteriophage and Its Current Status

Bacteriophage applications in aquaculture

Aquaculture has grown tremendously due to the big demand for its products. However, diseases affecting aquaculture and economic losses are worldwide problems and it needs low cost, sustainable, highly efficient, specific and eco-friendly therapeutants. Due to the rising up antibiotic resistant-microorganism, bacteriophage therapy has reinvigorated to replace antibiotics in agriculture, medicine, food safety and the environment. Likewise, it also holds great promise to avoid, control and treat bacteria in aquaculture to decrease the mortality level of different aquatic animal diseases. The isolation and characterization of new phages and phage application therapy to eliminate bacterial fish and shellfish pathogens such as Vibrio, Aeromonas, Pseudomonas, Lactococcus, Yersinia, Flavobacterium, and Streptococcus was gradually reported in aquaculture literature. The present review summarizes large-scale reports in vitro or in vivo use of aquaphage studies and applications in fish diseases from the 1980s to 2022 and future directions.

Engineered reporter phages for detection of Escherichia coli, Enterococcus, and Klebsiella in urine

The rapid detection and species-level differentiation of bacterial pathogens facilitates antibiotic stewardship and improves disease management. Here, we develop a rapid bacteriophage-based diagnostic assay to detect the most prevalent pathogens causing urinary tract infections: Escherichia coli, Enterococcus spp., and Klebsiella spp. For each uropathogen, two virulent phages were genetically engineered to express a nanoluciferase reporter gene upon host infection. Using 206 patient urine samples, reporter phage-induced bioluminescence was quantified to identify bacteriuria and the assay was benchmarked against conventional urinalysis. Overall, E. coli, Enterococcus spp., and Klebsiella spp. were each detected with high sensitivity (68%, 78%, 87%), specificity (99%, 99%, 99%), and accuracy (90%, 94%, 98%) at a resolution of ≥103 CFU/ml within 5 h. We further demonstrate how bioluminescence in urine can be used to predict phage antibacterial activity, demonstrating the future potential of reporter phages as companion diagnostics that guide patient-phage matching prior to therapeutic phage application.

Isolation of Clavibacter michiganensis subsp. michiganenesis-specific bacteriophages from tomato fields in Turkey and their biocontrol potential

BackgroundClavibacter michiganensis subsp. michiganensis (Cmm) is causing wilt and canker and significant economic losses in tomato production. Current control methods with conventional chemicals or antibiotics lost their effectiveness due to the development of bacterial resistance to this agent. Bacteriophages, viruses of bacteria, have been attracting increasing attention as an environmentally friendly means of controlling bacterial diseases. In this study, Cmm and Cmm-specific lytic bacteriophages were isolated from rhizosphere soil and phyllospheres of diseased tomato plants, and the biocontrol potential of bacteriophages was investigated.ResultsEight isolates that showed typical symptoms of Cmm infection in biochemical and pathogenicity tests contained the virulence gene pat-1 in the genome. Twenty-two native bacteriophages were isolated from 90 samples. As a result of the host range tests performed on the local Cmm isolates obtained in the study, temperature and pH tolerance tests were performed with ten phages with a wide host range. Phage24, Phage33 and Phage41, which have higher temperature and pH tolerance than other phages, were used to kill curve assay. Although the combined applications of phages were performed to increase efficacy, it was found that the application of Phage33 alone was more effective.ConclusionPhage33 may prove to be a suitable agent to be used as part of the microbial control strategy of Cmm. In further studies, Phage33 should be formulated to protect it from adverse environmental conditions and tested under field conditions to confirm the results obtained in this study.

Pheno- and Genotyping of Three Novel Bacteriophage Genera That Target a Wheat Phyllosphere Sphingomonas Genus.

Bacteriophages are viral agents that infect and replicate within bacterial cells. Despite the increasing importance of phage ecology, environmental phages-particularly those targeting phyllosphere-associated bacteria-remain underexplored, and current genomic databases lack high-quality phage genome sequences linked to specific environmentally important bacteria, such as the ubiquitous sphingomonads. Here, we isolated three novel phages from a Danish wastewater treatment facility. Notably, these phages are among the first discovered to target and regulate a Sphingomonas genus within the wheat phyllosphere microbiome. Two of the phages displayed a non-prolate Siphovirus morphotype and demonstrated a narrow host range when tested against additional Sphingomonas strains. Intergenomic studies revealed limited nucleotide sequence similarity within the isolated phage genomes and to publicly available metagenome data of their closest relatives. Particularly intriguing was the limited homology observed between the DNA polymerase encoding genes of the isolated phages and their closest relatives. Based on these findings, we propose three newly identified genera of viruses: Longusvirus carli, Vexovirus birtae, and Molestusvirus kimi, following the latest ICTV binomial nomenclature for virus species. These results contribute to our current understanding of phage genetic diversity in natural environments and hold promising implications for phage applications in phyllosphere microbiome manipulation strategies.

Structure, Biology, and Applications of Filamentous Bacteriophages.

The closely related Escherichia coli Ff filamentous phages (f1, fd, and M13) have taken a fantastic journey over the past 60 years, from the urban sewerage from which they were first isolated, to their use in high-end technologies in multiple fields. Their relatively small genome size, high titers, and the virions that tolerate fusion proteins make the Ffs an ideal system for phage display. Folding of the fusions in the oxidizing environment of the E. coli periplasm makes the Ff phages a platform that allows display of eukaryotic surface and secreted proteins, including antibodies. Resistance of the Ffs to a broad range of pH and detergents facilitates affinity screening in phage display, whereas the stability of the virions at ambient temperature makes them suitable for applications in material science and nanotechnology. Among filamentous phages, only the Ffs have been used in phage display technology, because of the most advanced state of knowledge about their biology and the various tools developed for E. coli as a cloning host for them. Filamentous phages have been thought to be a rather small group, infecting mostly Gram-negative bacteria. A recent discovery of more than 10 thousand diverse filamentous phages in bacteria and archaea, however, opens a fascinating prospect for novel applications. The main aim of this review is to give detailed biological and structural information to researchers embarking on phage display projects. The secondary aim is to discuss the yet-unresolved puzzles, as well as recent developments in filamentous phage biology, from a viewpoint of their impact on current and future applications.

Lytic Escherichia phage OSYSP acts additively and synergistically with gaseous ozone against Escherichia coli O157:H7 on spinach leaves

Bacteriophage and gaseous ozone are evolving as meritorious alternatives to conventional sanitizers in food postharvest applications. Here, we investigated the efficacy of sequential treatments of a lytic bacteriophage and gaseous ozone, during vacuum cooling of fresh produce, against Escherichia coli O157:H7. Spinach leaves were spot-inoculated with 105–107 CFU g−1 E. coli O157:H7 B6-914 and treated with Escherichia phage OSYSP spray (109 PFU g−1), gaseous ozone, or their combination. Vacuum cooling, which preceded or followed phage application but ran concomitantly with ozone treatment, was performed in a custom-made vessel at the following process sequence: vacuum to 28.5 in. Hg, vessel pressurization to 10 psig with gas containing 1.5 g ozone/kg gas-mix, holding for 30 min, and vessel depressurization to ambient pressure. Bacteriophage or gaseous ozone inactivated E. coli O157:H7, applied at different initial populations on spinach leaves, by 1.7–2.0 or 1.8–3.5 log CFU g−1, respectively. At the high inoculum levels tested (7.1 log CFU g−1), sequential treatments of phage and ozone reduced E. coli O157:H7 population by 4.0 log CFU g−1, but when treatment order was reversed (i.e., ozone followed by bacteriophage), the combination synergistically decreased pathogen’s population on spinach leaves by 5.2 log CFU g−1. Regardless the antibacterial application order, E. coli O157:H7 populations, applied initially at ~ 105 CFU g−1, were reduced below the enumeration method’s detection level (i.e., < 101 CFU g−1). The study proved that bacteriophage–ozone combination, applied in conjunction with vacuum cooling, is a potent pathogen intervention strategy in fresh produce post-harvest applications.

Phage Interactions with the Nervous System in Health and Disease.

The central nervous system manages all of our activities (e.g., direct thinking and decision-making processes). It receives information from the environment and responds to environmental stimuli. Bacterial viruses (bacteriophages, phages) are the most numerous structures occurring in the biosphere and are also found in the human organism. Therefore, understanding how phages may influence this system is of great importance and is the purpose of this review. We have focused on the effect of natural bacteriophages in the central nervous system, linking them to those present in the gut microbiota, creating the gut-brain axis network, as well as their interdependence. Importantly, based on the current knowledge in the field of phage application (e.g., intranasal) in the treatment of bacterial diseases associated with the brain and nervous system, bacteriophages may have significant therapeutic potential. Moreover, it was indicated that bacteriophages may influence cognitive processing. In addition, phages (via phage display technology) appear promising as a targeted therapeutic tool in the treatment of, among other things, brain cancers. The information collected and reviewed in this work indicates that phages and their impact on the nervous system is a fascinating and, so far, underexplored field. Therefore, the aim of this review is not only to summarize currently available information on the association of phages with the nervous system, but also to stimulate future studies that could pave the way for novel therapeutic approaches potentially useful in treating bacterial and non-bacterial neural diseases.