Administration Of Phages Research Articles

Bacteriophages as potential new mammalian pathogens

Increased intestinal permeability and translocation of gut bacteria trigger various polyaetiological diseases associated with chronic inflammation and underlie a variety of poorly treatable pathologies. Previous studies have established a primary role of the microbiota composition and intestinal permeability in such pathologies. Using a rat model, we examined the effects of exposure to a bacteriophage cocktail on intestinal permeability and relative abundance of taxonomic units in the gut bacterial community. There was an increase in markers of impaired gut permeability, such as the lactulose/mannitol ratio, plasma endotoxin concentrations, and serum levels of inflammation-related cytokines, following the bacteriophage challenge. We observed significant differences in the alpha diversity of faecal bacterial species and found that richness and diversity index values increased following the bacteriophage challenge. There was a reduction in the abundance of Blautia, Catenibacterium, Lactobacillus, and Faecalibacterium species and an increase in Butyrivibrio, Oscillospira and Ruminococcus after bacteriophage administration. These findings provide novel insights into the role of bacteriophages as potentially pathogenic for mammals and their possible implication in the development of diseases associated with increased intestinal permeability.

Efficacy of Phage Therapy in Controlling Rabbit Colibacillosis and Changes in Cecal Microbiota.

Phage therapy is a valid weapon that we could use to fight against pathogens. Bacteriophages kill bacteria and self-proliferate in the digestive tract. Furthermore, it was assumed that phage therapy could preserve the existing gut microbiota. In this study, 45 rabbits were equally divided into three groups after they were orally inoculated with pathogenic Escherichia coli to induce gut infection. Each group was treated with bacteriophage ZRP1 (Group P), ciprofloxacin lactate (Group A), or phosphate-buffered solution (PBS) (Group N). Another 15 healthy rabbits composed the control group (Group C). The body weight gain decreased significantly, but the white blood cell (WBC) count, especially the percentage of large WBCs, and the serum endotoxin levels increased significantly after infection. The result of microscopic examination of the ileum showed that E. coli ZR1 adhered to villi and caused hemorrhage inside the villi. Groups P and A rabbits recovered after treatments, and both bacteriophage and antibiotic treatment significantly decreased the eaeA gene concentration in cecal contents. The microbiota in cecal contents changed in infected rabbits that were treated with PBS. The relative abundance of Clostridiales and YS2 decreased but the relative abundance of Enterobacteriales increased significantly. According to the principal components analysis, the microbiota of Groups P and C rabbits were similar to one another in type and relative abundance but different from those of Groups N and A rabbits. The results demonstrated that oral administration of bacteriophage can cure gut infection with minimal impact on the cecal microbiota.

Impacts of Antibiotic and Bacteriophage Treatments on the Gut-Symbiont-Associated Blissus insularis (Hemiptera: Blissidae).

The Southern chinch bug, Blissus insularis, possesses specialized midgut crypts that harbor dense populations of the exocellular symbiont Burkholderia. Oral administration of antibiotics suppressed the gut symbionts in B. insularis and negatively impacted insect host fitness, as reflected by retarded development, smaller body size, and higher susceptibility to an insecticide, bifenthrin. Considering that the antibiotics probably had non-lethal but toxic effects on host fitness, attempts were conducted to reduce gut symbionts using bacteriophage treatment. Soil-lytic phages active against the cultures of specific Burkholderia ribotypes were successfully isolated using a soil enrichment protocol. Characterization of the BiBurk16MC_R phage determined its specificity to the Bi16MC_R_vitro ribotype and placed it within the family Podoviridae. Oral administration of phages to fifth-instar B. insularis, inoculated with Bi16MC_R_vitro as neonates had no deleterious effects on host fitness. However, the ingested phages failed to impact the crypt-associated Burkholderia. The observed inactivity of the phage was likely due to the blockage of the connection between the anterior and posterior midgut regions. These findings suggest that the initial colonization by Burkholderia programs the ontogeny of the midgut, providing a sheltered residence protected from microbial antagonists.

Prophylactic Bacteriophage Administration More Effective than Post-infection Administration in Reducing Salmonella enterica serovar Enteritidis Shedding in Quail.

Infections caused by Salmonella bacteria, often through poultry products, are a serious public health issue. Because of drawbacks associated with antibiotic prophylaxis, alternative treatments are sought. Bacterial viruses (bacteriophages) may provide an effective alternative, but concerns remain with respect to bacteriophage stability and effectiveness. To this end, we assessed the stability of a novel bacteriophage isolated from poultry excreta, siphovirus PSE, and its effectiveness in reducing Salmonella enterica serovar Enteritidis colonization in vitro and in vivo. Moreover, we sought to determine how the timing (prophylactic or therapeutic) and route (oral gavage or vent lip) of PSE administration impacted its effectiveness. Here we report that significant quantities of viable PSE bacteriophages were recovered following exposure to high and low pH, high temperatures, and bile salts, testifying to its ability to survive extreme conditions. In addition, we found that ileal lactic acid bacteria and Streptococcus spp. counts increased, but colibacilli and total aerobe counts decreased, in quail receiving phage PSE through both oral gavage and vent lip routes. In other experiments, we assessed the efficiency of PSE administration, in both prophylactic and therapeutic contexts, via either oral gavage or vent lip administration, on S. Enteritidis colonization of quail cecal tonsils. Our results demonstrate that administration of PSE as a preventive agent could reduce the S. Enteritidis colonization more effectively than post-challenge administration. Furthermore, oral administration of PSE phage is a more effective prophylactic tool for reduction of S. Enteritidis shedding in poultry than is vent lip administration.

Cover Picture: Biotechnology Journal 5/2016

AbstractThis regular issue of BTJ includes articles on biocatalysis, biomaterials, and industrial biotechnology. The cover picture shows several aspects of bacteriophage therapy, including phages adsorbing to bacterial cell surfaces, commercial preparations Pyo‐ and Intesi‐phages that are currently produced and used in Georgia, and administration of phages by medical staff to a patient. Image is provided by Aleksandre Ujmajuridze, Jon Caplin, Nino Chanishvili, and Aidan Coffey authors of ”Silk route to the acceptance and re‐implementation of bacteriophage therapy“ (http://dx.doi.org/10.1002/biot.201600023).

Corneal Infection Therapy with Topical Bacteriophage Administration.

Staphylococcus aureus is a major pathogen in bacterial keratitis, a vision-threatening disease. Although the incidence of S. aureus keratitis varies worldwide, the increasing trend of resistance to certain antibiotics makes this condition an important, global, healthcare concern. We report the case of a 65-year-old woman with nosocomial left-eye corneal abscess and interstitial keratitis.The patient then undergo topical Phage therapy with successful results.

Bacteriophage administration significantly reduces Shigella colonization and shedding by Shigella-challenged mice without deleterious side effects and distortions in the gut microbiota.

We used a mouse model to establish safety and efficacy of a bacteriophage cocktail, ShigActive™, in reducing fecal Shigella counts after oral challenge with a susceptible strain. Groups of inbred C57BL/6J mice challenged with Shigella sonnei strain S43-NalAcR were treated with a phage cocktail (ShigActive™) composed of 5 lytic Shigella bacteriophages and ampicillin. The treatments were administered (i) 1 h after, (ii) 3 h after, (iii) 1 h before and after, and (iv) 1 h before bacterial challenge. The treatment regimens elicited a 10- to 100-fold reduction in the CFU's of the challenge strain in fecal and cecum specimens compared to untreated control mice, (P < 0.05). ShigActiveTM treatment was at least as effective as treatment with ampicillin but had a significantly less impact on the gut microbiota. Long-term safety studies did not identify any side effects or distortions in overall gut microbiota associated with bacteriophage administration. Shigella phages may be therapeutically effective in a “classical phage therapy” approach, at least during the early stages after Shigella ingestion. Oral prophylactic “phagebiotic” administration of lytic bacteriophages may help to maintain a healthy gut microbiota by killing specifically targeted bacterial pathogens in the GI tract, without deleterious side effects and without altering the normal gut microbiota.

Bacteriophage-loaded nanostructured lipid carrier: improved pharmacokinetics mediates effective resolution of Klebsiella pneumoniae-induced lobar pneumonia.

This study examined the therapeutic and prophylactic potential of bacteriophages in a mouse model of Klebsiella pneumoniae lobar pneumonia. Phages were administered intraperitoneally. Liposome-entrapped phages (LP) were effective in treating infection, even when therapy was delayed by 3 days after the induction of pneumonia. In contrast, nonliposomal phages provided protection when administered 24 hours after infection. Administration of nonliposomal phages 6 hours prior to intranasal bacterial challenge resulted in complete protection, compared with LP, which was effective even when administered 48 hours prior to infection. Increased reduction and a greater increment in the levels of proinflammatory and antiinflammatory cytokines, respectively, in homogenates of lung from LP-treated mice were suggestive of increased efficacy of LP in the treatment of pneumonia. This is the first study to assess liposomes as a delivery vehicle for phage, and the results confirm the superiority of LP for both therapeutic and prophylactic applications.

이유자돈에서 Salmonella Typhimurium 감염에 대한 박테리오파지의 방어 효능

Salmonellosis has caused heavy losses in swine industry and implications for public health. Recently, the urgent problem of antibiotic resistance due to multidrug-resistant Salmonella spp. has been on the rise. The use of host-specific bateriophages as a biocontrol is one possible alternative. In this study, clinical signs, growth performance, quantification and detection of antigen, histopathological changes of gastrointestinal tracts were analyzed comparatively in weaned piglets according to administration of bacteriophages and challenge with Salmonella (S.) Typhimurium. Piglets challenged with S. Typhimurium after administered with bacteriophages showed reduced clinical signs, higher growth performance, lower bacterial shedding, lower quantificational value of antigens in intestines, higher V/C ratio and higher the number of goblet cells in intestines than piglets administered without bacteriophage and challenged with S. Typhimurium. These results indicate that feeding contained with bacteriophages has effect to prevent infection of S. Typhimurium in weaned piglets and suggest that a use of bacteriophage can be considered a valid antibiotic alternative.

Kingdom-Agnostic Metagenomics and the Importance of Complete Characterization of Enteric Microbial Communities

Advanced sequencing techniques have shown that bacteria are not the only complex and important microbes in the human intestine. Nonbacterial organisms, particularly the virome and the mycobiome, are important regulators of intestinal immunity and inflammation. The virome is mucosal and systemic; it can alter the host response to bacteria and interact with host genes and bacteria to contribute to disease pathogenesis. The human mycobiome is also complex and can contribute to intestinal inflammation. We review what has recently been learned about the nonbacterial and nonarchaeal microbes in the gastrointestinal tract, discussing their potential effects on health and disease and analytical approaches for their study. Studies of associations between the microbiome and intestinal pathology should incorporate kingdom-agnostic approaches if we are to fully understand intestinal health and disease.

The effect of prepartum intravaginal bacteriophage administration on the incidence of retained placenta and metritis

The objective of this study was to evaluate the effect of prepartum intravaginal administration of an anti-Escherichia coli bacteriophage cocktail on uterine health and reproductive performance of lactating dairy cows. The study was conducted on a commercial dairy farm located near Ithaca, New York, from March to September 2012, and 107 cows were enrolled. The cows were divided randomly into 2 groups (treatment and control); treatment group cows were submitted to 3 intravaginal administrations of 20mL of a bacteriophage cocktail including 10 different phages in a lysate with titration of approximately 109 plaque-forming units, on d 230, 260, and 275 of gestation. Control cows were not treated with a placebo, and physical examination and sample collection was conducted equally for both groups. Vaginal swabs were collected at d 230, 260, and 275d of gestation, cervical swabs were collected at 3±1d in milk, and blood was collected at 275d of gestation for nonesterified fatty acids and interleukin (IL)-6 serum level measurements and at 10 DIM for β-hydroxybutyrate and IL-6 serum level measurements. The bacteriophage cocktail significantly increased the incidence of retained placenta and metritis. Swab samples were cultured for E. coli and no difference was found between treatment and control groups. The lysate contained 6×109 IU of lipopolysaccharide/mL and could have caused prepartum overstimulation of toll-like receptor-4 (TLR4)-chemokine pathway, leading to a weaker immune response. However, IL-6 was not affected by treatment, although sample size was insufficient to prove a treatment effect on the suppression of the chemokine cascade and innate immune system. Bacteriophage treatment had an excellent bactericidal effect in previous in vitro trials; however, special attention must be given to production of the cocktail to obtain a purified solution free of E. coli bacterial products and fragments, particularly lipopolysaccharide.

Method of administration affects the ability of bacteriophage to prevent colibacillosis in 1-day-old broiler chickens

Bacteriophages are viruses that kill bacteria. They are plentiful in nature with no known activity in human or animal cells, making them an attractive alternative to antibiotics. The objective of this research was to determine if a coarse or a fine spray of bacteriophage would prevent colibacillosis induced by an intratracheal (IT) challenge with Escherichia coli. Two studies were conducted with 6 treatments: untreated control, birds treated with a spray administration of bacteriophage and not challenged, birds administered bacteriophage IT and not challenged, birds not treated and challenged IT with E. coli, birds sprayed with bacteriophage and IT challenged with E. coli, and birds administered bacteriophage IT and challenged IT with E. coli. There were 3 replicate pens of 10 birds per pen, per treatment, and all treatments were administered at 1 d of age. Study 1 was concluded when the birds were 19 d of age, and study 2 was concluded when the birds were 21 d of age. In both studies, neither a coarse nor a fine spray protected the birds from an IT E. coli challenge; however, when bacteriophage was administered IT there was complete protection. This research demonstrates the necessity for the administration of bacteriophage therapeutics to deliver high bacteriophage titers to the site of a bacterial infection.

Phage survival: The biodegradability of M13 phage display library in vitro

Administration of bacteriophages is used for phage therapy modulation of gut microbiome or for in vivo phage display. The aim of the study was to analyze the survival of M13 phage in different body fluids and tissues in vitro. The survival of M13 phage was measured in vitro in human blood, saliva, urine, artificial gastric juice (AGJ), and mouse homogenates of stomach, jejunum, and colon after defined time points (5, 15, or 45Min). The plates were inspected after overnight incubation and the plaques were counted. No phage was recovered after 5Min of incubation with AGJ. In urine, the phage survival was decreased by 44% after 5Min of incubation (P=0.004). In saliva, the recovered titer was decreased by 33% and 88% (P<0.05) after 15 and 45 Min, respectively. Phage coincubation with jejunum homogenate led to significant decrease of phage titer by 72% (P<0.01) after 15Min and by 99% (P<0.001) after 45Min. Decreased survival of M13 phage depending on time of incubation was proved under several in vitro conditions, with low pH in the AGJ having the most detrimental effect on phage survival. Phage pharmacokinetics described in vitro might have applications for the use of bacteriophages in vivo.

The effect of intrauterine administration of mannose or bacteriophage on uterine health and fertility of dairy cows with special focus on Escherichia coli and Arcanobacterium pyogenes

The objective of this study was to evaluate the effects of intrauterine administration of 50g of ultrapure mannose or a bacteriophage cocktail and the presence of Escherichia coli and Arcanobacterium pyogenes in the uterine lumen on uterine health and reproductive performance of lactating dairy cows. The study was conducted on a commercial dairy farm located near Ithaca, New York, from May 4 to January 20, 2011, and 597 cows were enrolled. The cows were divided randomly into 3 treatment groups, and treatments were administered at 2±1 d in milk (DIM). Treatment 1 consisted of intrauterine administration of 50g of ultrapure mannose powder divided in 4 pills; treatment 2 consisted of intrauterine administration of a bacteriophage cocktail that included 4 different phages in one pill, with a dose of approximately 107 plaque-forming units; and treatment 3 consisted of intrauterine administration of one empty pill (control). Intrauterine fluid swabs were collected on day of treatment and at 10±3 DIM; uterine lavage samples were collected at 35±3 DIM. Swabs and uterine lavage samples were cultured for E. coli and A. pyogenes. The intrauterine administration of mannose and bacteriophage did not affect uterine health, reproduction performance, or outcome of intrauterine secretion cultures for E. coli and A. pyogenes. Prevalence of intrauterine E. coli at 2±1 DIM and A. pyogenes at 2±1, 10±3, and 35±3 were affected by retained placenta. Additionally, prevalence of intrauterine E. coli at 10±3 and A. pyogenes at 35±3 DIM were associated with metritis, and cows that were diagnosed with clinical endometritis at 35±3 DIM had greater prevalence of intrauterine E. coli at 2±1 DIM and A. pyogenes at 35±3 DIM. Furthermore, cows positive for E. coli at 2±1 and 10±3 DIM and for A. pyogenes at 10±3 DIM had 1.63, 2.34, and 1.54 increased odds of having metritis. Cows positive for A. pyogenes at 35±3 DIM and for E. coli at 2±1 DIM had 19.8 and 2.66 higher odds of being diagnosed with clinical endometritis, respectively. Additionally, cows negative for E. coli at 2±1 DIM were 1.39 times more likely to conceive than cows positive cows for E. coli.

Evaluation of oral administration of bacteriophages to neonatal calves: Phage survival and impact on fecal Escherichia coli

Diarrhea is an important cause of morbidity and mortality in neonatal calves. Several enteropathogens are associated with diarrhea in young calves, with Enterotoxigenic Escherichia coli (ETEC) infection being the most common type of colibacillosis. The rise of antibiotic resistance in a number of medically important bacterial pathogens has revived interest in the use of bacteriophages as anti-bacterial therapeutic agents. Here we describe the results of a randomized, placebo-controlled, double-blinded study designed to evaluate the effect of an oral bacteriophage cocktail on fecal E. coli colony-forming units (CFU). Ten calves were enrolled in the study to either the bacteriophage group or the placebo group for 22 feedings. Calves in the bacteriophage treatment group (n=6) received a total of 106 plaque-forming units (PFU) (volume=5ml) of each of four bacteriophages while the placebo group (n=4) received only phosphate-buffered saline (5ml). Fresh fecal samples and blood samples were collected daily from each calf and analyzed for bacterial count and presence of bacteriophage. E. coli-infecting phages were recovered from all phage-treated calves at concentrations of 10² to 10³ PFU per gram of rectal contents, but none was detected in serum. Phage treatment caused a reduction in fecal E. coli when compared to the control group: the mean log CFU for the placebo-treated group was 9.25 (SE=0.42) versus 9.11 (SE=0.34) for the phage-treated group, but the difference was not statistically significant.

Efficacy of Bacteriophage Therapy on Horizontal Transmission of Salmonella Gallinarum on Commercial Layer Chickens

A Salmonella Gallinarum (SG)-specific bacteriophage isolated from sewage effluent was used to prevent horizontal transmission of SG in commercial layer chickens. Six-week-old chickens, each challenged with 5 x 10(8) colony-forming units of SG, cohabited with contact chickens treated with 10(6) plaque-forming units/kg of bacteriophage, prepared in feed additives, for 7 days before, and 21 days after challenge with SG. Mortality was observed for 3 wk after challenge and SG was periodically reisolated from the liver, spleen, and cecum of chickens. SG re-isolation from organs was decreased and a significant (P < 0.05) reduction in mortality was observed in contact chickens treated with the bacteriophage, as compared to untreated contact chickens, indicating that bacteriophage administration in feed additives significantly prevented the horizontal transmission of SG. These results provide important insights into prevention and control strategies against SG infection and suggest that the use of bacteriophages may be a novel, safe, and effectively plausible alternative to antibiotics for the prevention of SG infection in poultry.

Topical treatment of Pseudomonas aeruginosa otitis of dogs with a bacteriophage mixture: A before/after clinical trial

In an evaluation of a bacteriophage treatment for infection, ten dogs were included with chronic Pseudomonas aeruginosa otitis. Each received, directly into the auditory canal of one ear, a single dose of a topical preparation containing approximately 1 × 10 5 plaque forming units (PFU) of each of 6 bacteriophage strains, active against P. aeruginosa. At the time of bacteriophage administration and 48 h later each dog's core temperature was taken, its ear was assigned a clinical score (higher = worse condition) and aural swabs were taken for bacteriophage and P. aeruginosa counts. Forty eight hours after treatment the clinical score and P. aeruginosa count of all ears had fallen (mean score fall: 30.1%, range 7.7–56.3%, p < 0.0001; mean count fall: 67%, range 29.4–96.8%, p < 0.001). The bacteriophage counts had risen from the administered dose (mean rise: 99.1-fold, range 2.8–433.3-fold). No treatment related inflammation or other adverse events were detected during the trial period. This is the first report of a veterinary clinical trial of a bacteriophage treatment of infection. The results show that administration of this topical bacteriophage mixture leads to lysis of P. aeruginosa in the ear without apparent toxicity and that it has potential to be a convenient and effective treatment for P. aeruginosa otitis in dogs.

Bacteriophage Administration Reduces the Concentration of Listeria monocytogenes in the Gastrointestinal Tract and Its Translocation to Spleen and Liver in Experimentally Infected Mice.

To investigate the efficacy of phage supplementation in reducing pathogen numbers, mice were treated via oral gavage with a Listeria monocytogenes phage preparation (designated ListShield) before being orally infected with L. monocytogenes. The concentrations of L. monocytogenes in the liver, spleen, and intestines were significantly lower (P < .05) in the phage-treated than in the control mice. Phage and antibiotic treatments were similarly effective in reducing the levels of L. monocytogenes in the internal organs of the infected mice. However, the significant weight loss detected in the control and antibiotic-treated groups was not observed in the infected, ListShield-treated mice. Long-term (90 days), biweekly treatment of uninfected mice with ListShield did not elicit detectable changes in the microbiota of their large intestines or deleterious changes in their health. Our data support the potential feasibility of using bacteriophages to control proliferation of L. monocytogenes in mice without affecting commensal microbiota composition.

Bacteriophages reduce Escherichia coli O157:H7 levels in experimentally inoculated sheep

Bacteriophage biocontrol has potential as a means of mitigating the prevalence of Escherichia coli O157:H7 in ruminants. The efficacy of oral administration of bacteriophages for reducing fecal shedding of E. coli O157:H7 by sheep was evaluated using 20 Canadian Arcott rams (50.0 ± 3.0) housed in four rooms (n = 5) in a contained facility. The rams had ad libitum access to drinking water and a pelleted barley-based total mixed ration, delivered once daily. Experimental treatments consisted of administration of E. coli O157:H7 (O157), E. coli O157:H7+bacteriophages (O157+phage), bacteriophages (phage), and control (CON). Oral inoculation of the rams with 109 CFU of a mixture of four nalidixic acid-resistant strains of E. coli O157:H7 was performed on day 0. A mixture of 1010 PFU of bacteriophages P5, P8 and P11 was administered on days -2, -1, 0, 6 and 7. Fecal samples collected on 14 occasions over 21 d were analyzed for E. coli O157:H7, total E. coli, total coliforms and bacteriophages. Sheep in treatment O157+phage shed fewer (P &lt; 0.05) E. coli O157:H7 than did sheep in treatment O157. Populations of total coliforms and total E. coli were similar (P &lt; 0.05) among treatments, implying that bacteriophage lysis of non-target E. coli and coliform bacteria in the gastrointestinal tract did not occur. Bacteriophage numbers declined rapidly over 21 d, which likely reduced the chance of collision between bacteria and bacteriophage. Oral administration of bacteriophages reduced shedding of E. coli O157:H7 by sheep, but a delivery system that would protect bacteriophages during passage through the intestine may increase the effectiveness of this strategy as well as allow phage to be administered in the feed.Key words: Escherichia coli O157:H7, bacteriophage, sheep, environment, coliforms

Bacteriophage Therapy: Exploiting Smaller Fleas

Although bacteriophages have been used for the treatment of patients with bacterial infections in some regions of the world for >9 decades, adequate clinical trials of the safety and efficacy of the treatment have not been reported. The increasing problem of antibiotic resistance has, however, rekindled interest in this approach to therapy. Although potentially significant obstacles to systemic administration of phages exist, topical and oral administration of phages and/or phage products, such as lysins, are feasible in the short term. In addition to exploitation of the effects of native phages and phage products, bioengineering of phages will allow directed specificity and their use as delivery systems for antimicrobial and antivirulence molecules. This brief overview of the history and status of phage therapy, along with speculation about its future, provides a background for understanding of this imminent therapeutic modality.