Phage Dose Research Articles

Phage Delivery Strategies for Biocontrolling Human, Animal, and Plant Bacterial Infections: State of the Art.

This review aims at presenting the main strategies that are currently available for the delivery of bacteriophages to combat bacterial infections in humans, animals, and plants. It can be seen that the main routes for phage delivery are topical, oral, systemic, and airways for humans. In animals, the topical and oral routes are the most used. To combat infections in plant species, spraying the plant's phyllosphere or drenching the soil are the most commonly used methods. In both phage therapy and biocontrol using phages, very promising results have been obtained so far. However, more experiments are needed to establish forms of treatment and phage doses, among other parameters. Furthermore, in general, there is a lack of specific standards for the use of phages to combat bacterial infections.

CRISPR-Cas in Pseudomonas aeruginosa provides transient population-level immunity against high phage exposures.

The prokaryotic adaptive immune system, CRISPR-Cas (clustered regularly interspaced short palindromic repeats; CRISPR-associated), requires the acquisition of spacer sequences that target invading mobile genetic elements such as phages. Previous work has identified ecological variables that drive the evolution of CRISPR-based immunity of the model organism Pseudomonas aeruginosa PA14 against its phage DMS3vir, resulting in rapid phage extinction. However, it is unclear if and how stable such acquired immunity is within bacterial populations, and how this depends on the environment. Here, we examine the dynamics of CRISPR spacer acquisition and loss over a 30-day evolution experiment and identify conditions that tip the balance between long-term maintenance of immunity versus invasion of alternative resistance strategies that support phage persistence. Specifically, we find that both the initial phage dose and reinfection frequencies determine whether or not acquired CRISPR immunity is maintained in the long term, and whether or not phage can coexist with the bacteria. At the population genetics level, emergence and loss of CRISPR immunity are associated with high levels of spacer diversity that subsequently decline due to invasion of bacteria carrying pilus-associated mutations. Together, these results provide high resolution of the dynamics of CRISPR immunity acquisition and loss and demonstrate that the cumulative phage burden determines the effectiveness of CRISPR over ecologically relevant timeframes.

Evaluation of the impact of repeated intravenous phage doses on mammalian host-phage interactions.

Phage therapy has shown great promise for the treatment of multidrug-resistant bacterial infections. However, the lack of a thorough and organized understanding of phage-body interactions has limited its clinical application. Here, we administered different purified phages (Salmonella phage SE_SZW1, Acinetobacter phage AB_SZ6, and Pseudomonas phage PA_LZ7) intravenously to healthy animals (rats and monkeys) to evaluate the phage-induced host responses and phage pharmacokinetics with different intravenous (IV) doses in healthy animals. The plasma and the organs were sampled after different IV doses to determine the phage biodistribution, phage-induced cytokines, and antibodies. The potential side effects of phages on animals were assessed. A non-compartment model revealed that the plasma phage titer gradually decreased over time following a single dose. Repeated doses resulted in a 2-3 Log10 decline of the plasma phage titer at 5 min compared to the first dose, regardless of the type of phage administered in rats. Host innate immune responses were activated including splenic enlargement following repeated doses. Phage-specific neutralization antibodies in animals receiving phages were detected. Similar results were obtained from monkeys. In conclusion, the mammalian bodies were well-tolerant to the administered phages. The animal responses to the phages and the phage biodistribution profiles could have a significant impact on the efficacy of phage therapy.IMPORTANCEPhage therapy has demonstrated potential in addressing multidrug-resistant bacterial infections. However, an insufficient understanding of phage-host interactions has impeded its broader clinical application. In our study, specific phages were administered intravenously (IV) to both rats and monkeys to elucidate phage-host interactions and evaluate phage pharmacokinetics (PK). Results revealed that with successive IV administrations, there was a decrease in plasma phage concentrations. Concurrently, these administrations elicited both innate and adaptive immune responses in the subjects. Notably, the observed immune responses and PK profiles exhibited variation contingent upon the phage type and the mammalian host. Despite these variations, the tested mammals exhibited a favorable tolerance to the IV-administered phages. This underscores the significance of comprehending these interactions for the optimization of phage therapy outcomes.

Comparative study on the effect of bacteriophage on MDR E.coli from Urinary tract infections

Abstract Introduction/Objective Background: Urinary tract infections (UTIs) associated with urethral catheters are the most common infections during hospitalization. Hospitalization's most common infections are urethral catheter-related UTIs. Bacteriophages infect bacteria exclusively. Phage therapy, especially in Eastern Europe, was widely used before antibiotics. Due to the overuse of antibiotics and the development of resistant bacteria, this therapy approach is regaining popularity. Methods/Case Report Subjects and methods: 2223 UAE-based UTI patients (4–68 years old) were examined. The isolated bacteria were identified biochemically and tested for antimicrobial susceptibility using Mueller-Hinton agar disk diffusion (Kirby Bauer's). TSA (Liofilchem, Italy) kept isolates at 4 °C.for each examination. Tryptic Soy agar (TSA; Liofilchem, Italy) was used to grow one isolated colony overnight at 37 °C. This culture was transferred aseptically to 10 mL of fresh TSB medium and grown overnight at 37 °C to 0.8 optical density. 10 ml CaCl2 in 100 ml LB homogenized EMIRATE sewage water. Overnight homogenates with 9 E. coli isolates—24 hours of 37°C incubation. Samples were centrifuged at 12,000x g for five minutes—a 0.45 mm Millipore (France) filter produced phage lysate. TSA (Liofilchem, Italy) was used as a culture medium, and the centrifuged supernatant as phage suspension for the double-layer method. After 37 °C, plaques were examined. Single-plaque isolations followed. EOP was measured on double-layer agar for bacteria with a clear lysis zone (Adams, 1959). calculated EOP. Phages tested individually. EZ1 and EZ2 were negatively stained with Na-phosphotungstate for morphotype. Results (if a Case Study enter NA) Results: This study found presumptive E. coli colonies in 478 of 2223 (21.5%) samples by plating pre-enriched samples on MacConkey. E. coli B2 and B4 were sensitive to Ciprofloxacin, but most strains were resistant to antibiotics. E. coli B3 was Metronidazole-sensitive. Individual E. coli strains were susceptible to phage infection. Podoviruses were the two phages. 3 log decreased due to E. coli growth. Conclusion This study shows that a single dose of phage can reduce pathogenic MDR E.coli. Phages' high bacterial inactivation efficiency, safety, and prolonged survival, even in urine samples, allow depth studies, especially in vivo studies, to control urinary tract infection and overcome E.coli resistance.

Slow growing bacteria survive bacteriophage in isolation

The interactions between bacteria and bacteriophage have important roles in the global ecosystem; in turn changes in environmental parameters affect the interactions between bacteria and phage. However, there is a lack of knowledge on whether clonal bacterial populations harbour different phenotypes that respond to phage in distinct ways and whether the abundance of such phenotypes within bacterial populations is affected by variations in environmental parameters. Here we study the impact of variations in nutrient availability, bacterial growth rate and phage abundance on the interactions between the phage T4 and individual Escherichia coli cells confined in spatial refuges. Surprisingly, we found that fast growing bacteria survive together with all of their clonal kin cells, whereas slow growing bacteria survive in isolation. We also discovered that the number of bacteria that survive in isolation decreases at increasing phage doses possibly due to lysis inhibition in the presence of secondary adsorptions. We further show that these changes in the phenotypic composition of the E. coli population have important consequences on the bacterial and phage population dynamics and should therefore be considered when investigating bacteria-phage interactions in ecological, health or food production settings in structured environments.

Reassessing the forgotten cure: Efficacy of bacteriophage as a therapeutic agent against MRSA in diabetic rats

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the leading causes of increased amputation, morbidity and mortality among patients with diabetic foot infections. These infections are refractory to antibiotics. Therefore, alternative therapy using phages has re-emerged as a promising option to treat such infections. The present study was conducted to evaluate the efficacy of phage in treating diabetic wound infection caused by MRSA 43300 in rats. Phages were isolated against MRSA 43300 and purified as per standard procedures. Diabetes was induced in healthy albino rats using streptozotocin. The minimum lethal dose (MLD) of MRSA and minimum protective dose (MPD) of phage was identified. A full-size excision wound was created on the back of rats. The rats were divided into five groups (Group I – Non-infected and untreated, Group II- Infected and untreated, Group III - Infected and phage challenged, Group IV- Infected and antibiotic treated, Group V – Non-infected and phage treated). Groups II, III and IV were infected with MLD of MRSA 43300. After 48 h of infection, Group III was treated with a single dose of MPD of phage and group IV was treated with mupirocin on all days. The wound were sampled on designated days, Gram stain was done to detect for the presence of pus cells and Gram positive cocci. Standard culture methods were used to confirm the presence of phage and MRSA 43300 and estimate their titre on different days among different groups. Wound contraction rate was recorded to compare the duration of wound healing between all the groups. Group II served as bacterial control in which all the rats died after 72h. The phage treated group III showed the presence of Gram positive cocci and pus cells in Gram stain till 6 days. Viable MRSA was isolated till 4 days post treatment and more than 50% of the wound contracted by day 12 of infection. Phage-treated group showed better wound contraction rate and faster bacterial clearance compared to antibiotic-treated group. The results of the present study help to reconsider phage therapy for treatment of drug-resistant infection.

"Two Is Better Than One": The Multifactorial Nature of Phage-Antibiotic Combinatorial Treatments Against ESKAPE-Induced Infections.

Phage-antibiotic synergy (PAS) has been extensively explored over the past decade, with the aim of developing more effective treatments against multidrug-resistant organisms. However, it remains unclear how to effectively combine these two approaches. To address this uncertainty, we assessed four main aspects of PAS interactions in this review, seeking to identify commonalities of combining treatments within and between bacterial species. We examined all literature on PAS efficacy toward ESKAPE pathogens and present an analysis of the data in papers focusing on: (1) order of treatment, (2) dose of both phage and antibiotics, (3) mechanism of action, and (4) viability of transfer from in vivo or animal model trials to clinical applications. Our analysis indicates that there is little consistency within phage-antibiotic therapy regimens, suggesting that highly individualized treatment regimens should be used. We propose a set of experimental studies to address these research gaps. We end our review with suggestions on how to improve studies on phage-antibiotic combination therapy to advance this field.

A bacteriophage cocktail delivered in feed significantly reduced Salmonella colonization in challenged broiler chickens

ABSTRACT Nontyphoidal Salmonella spp. are a leading cause of human gastrointestinal infections and are commonly transmitted via the consumption of contaminated meat. To limit the spread of Salmonella and other food-borne pathogens in the food chain, bacteriophage (phage) therapy could be used during rearing or pre-harvest stages of animal production. This study was conducted to determine if a phage cocktail delivered in feed is capable of reducing Salmonella colonization in experimentally challenged chickens and to determine the optimal phage dose. A total of 672 broilers were divided into six treatment groups T1 (no phage diet and unchallenged); T2 (phage diet 106 PFU/day); T3 (challenged group); T4 (phage diet 105 PFU/day and challenged); T5 (phage diet 106 PFU/day and challenged); and T6 (phage diet 107 PFU/day and challenged). The liquid phage cocktail was added to mash diet with ad libitum access available throughout the study. By day 42 (the concluding day of the study), no Salmonella was detected in faecal samples collected from group T4. Salmonella was isolated from a small number of pens in groups T5 (3/16) and T6 (2/16) at ∼4 × 102 CFU/g. In comparison, Salmonella was isolated from 7/16 pens in T3 at ∼3 × 104 CFU/g. Phage treatment at all three doses had a positive impact on growth performance in challenged birds with increased weight gains in comparison to challenged birds with no phage diet. We showed delivering phages via feed was effective at reducing Salmonella colonization in chickens and our study highlights phages offer a promising tool to target bacterial infections in poultry.

Administration of Bacteriophages via Nebulization during Mechanical Ventilation: In Vitro Study and Lung Deposition in Macaques.

Bacteriophages have been identified as a potential treatment option to treat lung infection in the context of antibiotic resistance. We performed a preclinical study to predict the efficacy of delivery of bacteriophages against Pseudomonas aeruginosa (PA) when administered via nebulization during mechanical ventilation (MV). We selected a mix of four anti-PA phages containing two Podoviridae and two Myoviridae, with a coverage of 87.8% (36/41) on an international PA reference panel. When administered via nebulization, a loss of 0.30-0.65 log of infective phage titers was measured. No difference between jet, ultrasonic and mesh nebulizers was observed in terms of loss of phage viability, but a higher output was measured with the mesh nebulizer. Interestingly, Myoviridae are significantly more sensitive to nebulization than Podoviridae since their long tail is much more prone to damage. Phage nebulization has been measured as compatible with humidified ventilation. Based on in vitro measurement, the lung deposition prediction of viable phage particles ranges from 6% to 26% of the phages loaded in the nebulizer. Further, 8% to 15% of lung deposition was measured by scintigraphy in three macaques. A phage dose of 1 × 109 PFU/mL nebulized by the mesh nebulizer during MV predicts an efficient dose in the lung against PA, comparable with the dose chosen to define the susceptibility of the strain.

Phage-host-immune system dynamics in bacteriophage therapy: basic principles and mathematical models.

Phage therapy is progressively being recognized as a viable alternative to conventional antibiotic treatments, particularly in the context of multi-drug resistant bacterial challenges. However, the intricacies of the pharmacokinetics and pharmacodynamics (PKPD) pertaining to phages remain inadequately elucidated. A salient characteristic of phage PKPD is the inherent ability of phages to undergo replication. In this review, I proffer mathematical models that delineate the intricate dynamics encompassing the phage, the host organism, and the immune system. Fundamental tenets associated with proliferative and inundation thresholds are explored, and distinctions between active and passive therapies are accentuated. Furthermore, I present models that aim to illuminate the multifaceted interactions amongst diverse phage strains and bacterial subpopulations, each possessing distinct sensitivities to phages. The synergistic relationship between phages and the immune system is critically examined, demonstrating how the host's immunological function can influence the requisite phage dose for an optimal therapeutic outcome. A profound understanding of the presented modeling methodologies is paramount for researchers in the realms of clinical pharmacology and PKPD modeling interested in phage therapy. Such insights facilitate a more nuanced interpretation of dose-response relationships, enable the selection of potent phages, and aid in the optimization of phage cocktails.

Safety and microbiological activity of phage therapy in persons with cystic fibrosis colonized with Pseudomonas aeruginosa: study protocol for a phase 1b/2, multicenter, randomized, double-blind, placebo-controlled trial

BackgroundBacteriophages (phages) are a promising anti-infective option for human disease. Major gaps remain in understanding their potential utility.MethodsThis is a randomized, placebo-controlled, double-blind study of a single dose of intravenous phage in approximately 72 clinically stable adult cystic fibrosis volunteers recruited from up to 20 US sites with Pseudomonas aeruginosa airway colonization. The single dose of phage consists of a mixture of four anti-pseudomonal phages. Six sentinel participants will be sequentially enrolled with dose escalation of the phage mixture by one log10 beginning with 4 × 107 plaque-forming units in an unblinded stage 1. If no serious adverse events related to the study product are identified, the trial will proceed to a double-blinded stage 2. In stage 2a, 32 participants will be randomly assigned to one of three phage dosages or placebo in a 1:1:1:1 allocation. An interim analysis will be performed to determine the phage dosage with the most favorable safety and microbiological activity profile to inform phage dosing in stage 2b. During stage 2b, up to 32 additional volunteers will be randomized 1:1 to the phage or placebo arm. Primary outcomes include (1) the number of grade 2 or higher treatment-emergent adverse events, (2) change in log10P. aeruginosa total colony counts in sputum, and (3) the probability of a randomly selected subject having a more favorable outcome ranking if assigned to receive phage therapy versus placebo. Exploratory outcomes include (1) sputum and serum phage pharmacokinetics, (2) the impact of phage on lung function, (3) the proportion of P. aeruginosa isolates susceptible to the phage mixture before and after study product administration, and (4) changes in quality of life.DiscussionThis trial will investigate the activity of phages in reducing P. aeruginosa colony counts and provide insights into the safety profile of phage therapy.Trial registrationClinicalTrials.gov NCT05453578. Registered on 12 July 2022.

Bactericidal Synergism between Phage YC#06 and Antibiotics: a Combination Strategy to Target Multidrug-Resistant Acinetobacter baumannii In Vitro and In Vivo.

ABSTRACTPhage-antibiotic combination (PAC) therapy is a potential new alternative to treat infections caused by pathogenic bacteria, particularly those caused by antibiotic-resistant bacteria. In the present study, phage YC#06 against highly multidrug-resistant Acinetobacter baumannii 4015 was isolated, identified, and characterized. Compared with antibiotics alone, the time-kill experiments in vitro showed that YC#06 and antibiotic mixtures that include the chloramphenicol, imipenem, and cefotaxime combination could produce phage-antibiotic synergy (PAS), which reduced the ultimate effective concentration of antibiotics. No phage-resistant bacteria have been isolated during the whole time-kill experiments in vitro. Of note, PAS was dose dependent, requiring a moderate phage dose to achieve maximum PAS effect. In addition, PAS could effectively inhibit biofilm formation and remove mature biofilms in vitro. Furthermore, PAS between the combination of YC#06 and antibiotic mixtures in vivo was validated using a zebrafish infection model. Overall, the results of this study demonstrate that PAC could be a viable strategy to treat infection caused by high-level multidrug-resistant Acinetobacter baumannii or other drug-resistant bacteria through switching to other types of phage and antibiotic mixtures.IMPORTANCE The treatment of multidrug-resistant bacterial infection is an urgent clinical problem. The combination of bacteriophages and antibiotics could produce synergistic bactericidal effects, which could reduce the emergence of antibiotic resistance and antibiotic consumption in antibiotic-sensitive bacteria, restore efficacy to antibiotics in antibiotic-resistant bacteria, and prevent the occurrence of phage-resistant bacteria. Phage-antibiotic combination (PAC) might be a potential new alternative for clinical treatment of multidrug-resistant bacterial infections.

The comparison of lytic activity of isolated phage and commercial Intesti bacteriophage on ESBL producer E. coli and determination of Ec_P6 phage efficacy with in vivo Galleria mellonella larvae model

Antibiotic resistance is one of the crucial public health challenges. As a result of rising resistance, as an alternative to antimicrobials, demands for bacteriophage therapy have increased significantly over the years. The objective of this study was to isolate and characterize potentially therapeutic phages active against Escherichia coli (E. coli) and compare the efficacy with commercial Intesti bacteriophage on the extended-spectrum beta-lactamase (ESBL) positive E. coli (ESBL-EC) and performed the effectiveness of bacteriophage using the Galleria mellonella (G. mellonella) larvae model. Intesti bacteriophage is a polyvalent bacteriophage-based drug. The isolated bacteriophages were obtained from the river and clinical isolates of E. coli were used for the enrichment of bacteriophage isolation. The phages were first screened based on plaque morphology and host ranges determined on clinical strains. The susceptibility of phages was determined against 50 clinical isolates of E. coli and eight different laboratory isolates using the spot test technique. E. coli lytic phage Ec_P6 was used to determine the therapeutic and preventive effects on the G. mellonella larvae model. The slides were prepared by G. mellonella hemolymph for cytologic examination, stained with May Grünwald Giemsa (MGG), and evaluated by light microscopy. The results of the activities revealed lytic spectra ranging from 24% to 97%. Overall strains were susceptible to one or more phages from the panel. It was proved that Intesti bacteriophage is very effective in a wide variety of strains of E. coli including test strains, also showed that isolated Ec_P6 phage is as effective as commercial phage. The best MOI of this phage was 0.01, and infectivity decreased above 60 °C. The results suggest that phage is stable at pH values ranging between 5.0 and 9.0. In vivo study was found that in E. coli infection to achieve a survival high rate the infected larvae should be after 2 h treated with 0.01 MOI phage (10 μL, 106 PFU/mL) and colistin doses (10 μL, 2.5 mg/kg). It also prevented infection, increasing the survival of the larvae compared to the untreated control group. Ec_P6 phage was found to have a potential for the treatment of E. coli infections.

The effects of different doses of inhaled bacteriophage therapy for Pseudomonas aeruginosa pulmonary infections in mice

ObjectivesInhaled phage therapy has been revisited as a potential treatment option for respiratory infections caused by multidrug-resistant Pseudomonas aeruginosa; however, there is a distinct gap in understanding the dose–response effect. The aim of this study was to investigate the dose–response effect of Pseudomonas-targeting phage PEV31 delivered by the pulmonary route in a mouse lung infection model. MethodsNeutropenic BALB/c mice were infected with multidrug-resistant P. aeruginosa (2 × 104 colony-forming units) through the intratracheal route and then treated with PEV31 at three different doses of 7.5 × 104 (Group A), 5 × 106 (Group B), and 5 × 108 (Group C) plaque-forming units, or phosphate-buffered saline at 2 hours postinoculation. Mice (n = 5–7) were euthanized at 2 hours and 24 hours postinfection, and lungs, kidneys, spleen, liver, bronchoalveolar lavage fluid, and blood were collected for bacteria and phage enumeration. ResultsAt 24 hours postinfection, all phage-treated groups exhibited a significant reduction in pulmonary bacterial load by 1.3–1.9 log10, independent of the delivered phage dose. The extent of phage replication was negatively correlated with the dose administered, with log10 titre increases of 6.2, 2.7, and 9 for Groups A, B, and C, respectively. Phage-resistant bacterial subpopulations in the lung homogenate samples harvested at 24 hours postinfection increased with the treatment dose (i.e. 30%, 74%, and 91% in respective Groups A–C). However, the mutants showed increased susceptibility to ciprofloxacin, impaired twitching motility, and reduced blue-green pigment production. The expression of the inflammatory cytokines (IL-1ß and IL-6, and TNF-α) was suppressed with increasing PEV31 treatment dose. DiscussionThis study provides the dose–response effect of inhaled phage therapy that may guide dose selection for treating P. aeruginosa respiratory infections in humans.

Gastrointestinal Dynamics of Non-Encapsulated and Microencapsulated Salmonella Bacteriophages in Broiler Production.

Simple SummaryBacteriophages are viruses that kill targeted bacteria and could be used as a therapy against multidrug-resistant bacteria in animal production. Gastrointestinal tract conditions throughout the broiler production cycle might compromise the efficacy of bacteriophage oral administration against Salmonella. Microencapsulation of phages could protect and prevent the premature release of the bacteriophage, thereby allowing targeted delivery to the colonization site of Salmonella, the caecum. This study was designed to assess the optimal timing of the phage intervention over a 42-day production cycle and to compare microencapsulated (delivered in animal feed) and non-encapsulated phages (delivered through the drinking water) delivery along the gastrointestinal tract. Results of this study suggest that microencapsulation of the phages in a Eudragit® L100 pH-responsive formulation allowed targeted delivery of the phage to the chicken caecum. Microencapsulation of phages administered orally through animal feed could be a promising method to control Salmonella in the field at any time during the animal rearing period.Bacteriophage therapy is being considered as a promising tool to control Salmonella in poultry. Nevertheless, changes in gastrointestinal tract environmental conditions throughout the production cycle could compromise the efficacy of phages administered orally. The main objectives of this study were to assess the optimal timing of the phage administration over a 42-day production cycle and to compare microencapsulated and non-encapsulated phages and the spatial and temporal dynamics of the phage delivery along the gastrointestinal tract. Phage FGS011 was encapsulated in the pH-responsive polymer Eudragit® L100 using the process of spray drying. At different weeks of the chicken rearing period, 15 broilers were divided into three groups. Over a period of 24 h, group 1 received non-encapsulated phages (delivered through drinking water), group 2 received microencapsulated phages (incorporated in animal feed), and group 3 did not receive any phages. Microencapsulation was shown to enable efficient delivery of the bacteriophages to the animal gut and cecum throughout the animal rearing period. During the six weeks of application, the crop displayed the highest phage concentration for both phage delivery methods. The L100 based encapsulation offered significant protection to the phages from the harsh environmental conditions in the PV-Gizzard (not seen with phages administered in drinking water) which may help in the delivery of high phage doses to the cecum. Future Salmonella challenge studies are necessary to demonstrate the benefits of microencapsulation of phages using L100 formulation on phage therapy in field studies during the rearing period.

Bacteriophage mediated control of necrotic enteritis caused by C. perfringens in broiler chickens.

In Egypt, little attention has been paid to the isolation and application of C. perfringens phages for treating necrotic enteritis at the farm level. This study aims to evaluate the efficiency of the podovirus C. perfringens phage in treating necrotic enteritis in broiler chickens. Accordingly, C. perfringens phage was isolated from cecal samples of apparently healthy chickens and characterized by transmission electron microscopy, thermal stability test, and pH stability test. Commercial 14-day-old Arbor Acres broiler chickens were allocated to three groups: group Ӏ received BHI broth and assigned as a negative control, group П served as a positive control group that was challenged with C. perfringens via oral gavage for four successive days, and group Ш was administrated six phage doses on several occasions after oral gavage challenge with C. perfringens. Daily clinical symptoms and mortality were recorded. At three-time intervals, necrotic enteritis lesions were scored. Cecal samples were examined for re-isolation and counting of C. perfringens. The isolated C. perfringens phage was a podovirus with an icosahedral head diameter of 78.7nm and a short non-contractile tail length of 22.2nm. It remained stable for 60min at 30°C and 50°C at pH values of 2, 4, 8, and 10. The phage-treated group (Ш) showed mild gross lesions with a lower mortality rate and reduced colony-forming units than the positive control group (П). The findings revealed that the isolated C. perfringens phage effectively treated experimental necrotic enteritis in broiler chickens.

The interactions of bacteriophage Ace and Shiga toxin-producing Escherichia coli during biocontrol.

Strictly lytic phages are considered powerful tools for biocontrol of foodborne pathogens. Safety issues needed to be addressed for the biocontrol of Shiga toxin-producing Escherichia coli (STEC) include: lysogenic conversion, Shiga toxin production through phage induction, and emergence/proliferation of bacteriophage insensitive mutants (BIMs). To address these issues, two new lytic phages, vB_EcoS_Ace (Ace) and vB_EcoM_Shy (Shy), were isolated and characterized for life cycle, genome sequence and annotation, pH stability and efficacy at controlling STEC growth. Ace was efficient in controlling host planktonic cells and did not stimulate the production of the Stx prophage or Shiga toxin. A single dose of phage did not lead to the selection of BIMs. However, when reintroduced, BIMs were detected after 24 h of incubation. The gain of resistance was associated with lower virulence, as a subset of BIMs failed to agglutinate with O157-specific antibody and were more sensitive to human serum complement. BIM's biofilm formation capacity and susceptibility to disinfectants was equal to that of the wild-type strain. Overall, this work demonstrated that phage Ace is a safe biocontrol agent against STEC contamination and that the burden of BIM emergence did not represent a greater risk in environmental persistence and human pathogenicity.

Protective efficacy of phage PVN02 against haemorrhagic septicaemia in striped catfish Pangasianodon hypophthalmus via oral administration.

Haemorrhagic septicaemia caused by Aeromonas hydrophila in striped catfish (Pangasianodon hypophthalmus) is one of the most important aquatic diseases in the Mekong Delta, Vietnam. However, antibiotic-resistant A.hydrophila strains have become popular and resulted in inadequate control of the disease in striped catfish farms. This study investigates the protective efficacy of bacteriophage PVN02 against haemorrhagic septicaemia in striped catfish via oral administration. The phage-containing pellets were prepared by spraying the phage solution on food pellets at 20ml/kg. The rate of phage desorption from the food pellets into the water was very low; the phage titres in the water were approximately log 1.0 PFU/ml or undetectable. The in vivo experiment evaluating the protective efficacy of PVN02 against haemorrhagic septicaemia in striped catfish was conducted using 21 groups of 1,260 fish in 50-L plastic tanks in triplicate. The catfish were fed twice daily with phage-sprayed pellets. Different densities of bacterial suspensions were added into the tanks for 24hr. Without the existence of the phage, the highest mortality rate was 68.3±2.9% at the highest density of bacterial suspension. In contrast, the mortality rate at the highest density of bacterial suspension was significantly reduced to 8.33±2.9% or 16.67±2.9% at the phage dose of log 6.2±0.09 or log 4.2±0.09 PFU/g. This study provides a very practical manner of applying phage therapy to prevent disease in large-scale striped catfish farms.

Bacteriophage Encapsulation Using Spray Drying for Phage Therapy.

Exploiting the potential of bacteriophages for phage therapy is an exciting future prospect. However, in order to be successful, there is a pressing need for the manufacture of safe and efficacious phage drug products to treat patients. Scalable manufacture of phage biologics as a stable solid dry powder form is highly desirable and achievable using the process of spray drying. Spray drying of purified phage suspensions formulated with suitable excipients can be carried out in a single step with high process throughput and at relatively low cost. The resulting phage-containing powders can possess good storage shelf-life. The process allows control over the final phage dose in the powder and production of microparticles suitable for a variety of therapeutic uses. Spray dried powders may include different polymer formulations employing a multitude of different triggers for phage release at the target site including pH, enzymes, virulence factors etc. The activity of the phages in spray dried powders is adversely affected during spray drying due to dessication and thermal stresses which need to be controlled. The choice of polymers, excipients and moisture content of the dry powders affects the material glass transition temperature and the stability of the phages during storage. The storage temperature and storage humidty are important factors affecting the stability of the phages in the dry powders. A quality by design (QbD) approach for phage drug product development needs to identify drug product characteristics that are critical to quality from the patient's perspective and translates them into the critical quality attributes (CQA) of the drug product. The relationship between the phage drug product CQAs and formulation development and spray drying process conditions are discussed in this article.

Potential of a Bacteriophage Isolated from Wastewater in Treatment of Lobar Pneumonia Infection Induced by Klebsiella pneumoniae in Mice.

The potential of bacteriophages as alternative treatment for multidrug-resistant (MDR) Klebsiella pneumoniae-related infections has recently gained much interest. The purpose of this research was to isolate and characterize a K. pneumoniae-specific lytic phage with the potential to treat experimental lobar pneumonia induced by K. pneumoniae in mice. A lytic phage was isolated from an urban wastewater sample in Tehran and characterized by transmission electron microscopy (TEM), thermal, pH, and chloroform stability before being employed for treatment of mice infected with K. pneumoniae in an experimental model of lobar pneumonia. BALB/C mice were challenged by intranasal inoculation with 108 colony-forming units (CFU/ml) of K. pneumoniae ATCC10031 followed by an intraperitoneal injection of the isolated phage using 1010 and 109 plaque-forming units (PFU/ml) simultaneously or 24h post infection. Control groups of mice received bacteria or bacteriophage alone. Mice were euthanized daily up to 7days post infection and examined for abnormality in their lungs and livers followed by determining the number of phages and bacteria in plasma and lung homogenates. The isolated phage (vB_KpnM-Teh.1) belonged to the Myoviridae family, was stable at 37°C, pH 7, and was resistant to chloroform. Treatment of mice with a single dose of phage simultaneously at the time of infection, or 24h post infection, resulted in seven and five logs decrease of CFU/ml in the lung homogenates up to 3days after phage administration, respectively. The isolated phage may have the potential as a therapeutic agent against K. pneumoniae infections.