Phage-treated Group Research Articles

A novel K57-specific Klebsiella pneumoniae phage disinfects milk and inhibits biofilm formation

Klebsiella pneumoniae (K. pneumoniae), especially serotype K57, is a major pathogen in dairy bovine mastitis, causing significant economic losses and posing a public health risk through contaminated dairy products. In addition, K57 is highly virulent among serotypes infecting humans, leading to serious conditions such as pyogenic liver abscess. In this study, we isolated and identified a bacteriophage called vB_Kp_Z57 that targets K57 serotype K. pneumoniae isolates from human and bovine sources. The phage has a latent period of 4 min and can produce approximately 233 phages per infected bacterial cell. vB_Kp_Z57 is stable over a temperature range of 4 °C to 60 °C and a pH range of 5–11. Sequencing and assembly of the phage genome revealed DNA molecules consisting of 40,316 base pairs. After genome annotation with bioinformatics software, it was discovered that the phage genome encodes 48 proteins, with no detected lysogeny genes, toxin genes, or tRNA-related genes. vB_Kp_Z57 was classified as a member of genus Przondovirus, family Autographiviridae. The results of bacteriostatic experiments show that vB_Kp_Z57 has a good effect on inhibiting bacterial growth. Notably, vB_Kp_Z57 significantly inhibited the growth of its host bacteria in milk at refrigerated temperatures of 4 °C and room temperature. Biofilm inhibition experiments revealed that phage vB_Kp_Z57 significantly reduced OD595 values in all phage-treated groups, indicating its potential as an effective biocontrol agent against K. pneumoniae. Our findings show the promise of vB_Kp_Z57 as a novel and environmentally friendly solution to address the challenges associated with K. pneumoniae in dairy production.

Lining the small intestine with mycobacteriophages protects from Mycobacterium avium subsp. paratuberculosis and eliminates fecal shedding

Johne's disease (JD), a chronic, infectious enteritis caused by Mycobacterium avium subsp. paratuberculosis (MAP), affects wild and domestic ruminants. There is no cure or effective prevention, and current vaccines have substantial limitations, leaving this disease widespread in all substantial dairy industries causing economic, and animal welfare implications. Mycobacteriophages (MPs) have been gaining interest in recent years and are proposed as a promising solution to curtailing MAP infection. Using a well-validated infection model, we have demonstrated the preventative potential of MPs to protect dairy calves against MAP infection. Calves were supplemented daily with a phage cocktail from birth till weaning at 2 m of age and inoculated with MAP at 2 wk of age. Infection status was measured for 4.5 mo through blood, fecal, and postmortem tissue samples. Our findings highlight the remarkable efficacy of orally administered MPs. Notably, fecal shedding of MAP was entirely eliminated within 10 wk, in contrast to the infected control group where shedding continued for the entirety of the trial period. Postmortem tissue culture analysis further supported the effectiveness of MPs, with only 1 out of 6 animals in the phage-treated group testing positive for MAP colonized tissues compared to 6 out of 6 animals in the infected control group. Additionally, plaque assay results demonstrated the ability of phages to persist within the intestinal tract. Collectively, these results underscore the potential of orally administered MP cocktails as a highly effective intervention strategy to combat JD in dairy calves and by extension in the dairy industry.

Evaluating the efficacy of a phage cocktail against Pseudomonas fluorescens group strains in raw milk: microbiological, physical, and chemical analyses.

The objective of this study was to investigate the effectiveness of a phage cocktail against Pseudomonas fluorescens group and its effect on the microbial, physical and chemical properties of raw milk during different storage conditions. A phage cocktail consisting of Pseudomonas fluorescens, Pseudomonas tolaasii, and Pseudomonas libanensis phages was prepared. As a result, reductions in fluorescent Pseudomonas counts of up to 3.44 log units for the storage at 4°C and 2.38 log units for the storage at 25°C were achieved. Following the phage application, it is found that there was no significant difference in the total mesophilic aerobic bacteria and Enterobacteriaceae counts. However, it was observed that the number of lactic acid bacteria was higher in phage-treated groups. The results also showed that pH values in the phage added groups were lower than the others and the highest titratable acidity was obtained only in the bacteria-inoculated group. As a future perspective, this study suggests that, while keeping the number of target microorganisms under control in the milk with the use of phages during storage, the microbiota and accordingly the quality parameters of the milk can be affected. This work contributes to the development of effective strategies for maintaining the quality and extending the shelf life of milk and dairy products.

Phage cocktail administration to reduce Salmonella load in broilers

Salmonella is a serious foodborne pathogen that can cause gastrointestinal disease through the consumption of contaminated foods; including poultry meat. Salmonella is commonly present in the intestinal tract of poultry and farm environments, posing a potential risk of contamination during the processing of poultry meat. This study was a continuation in evaluating the effects of our previously developed phage cocktail targeting Salmonella at large-scale trials in commercial broiler farms, in which this cocktail considerably lowered Salmonella colonization in the gut of broilers. The phage cocktail given to broilers showed resistance to temperatures of up to 65 °C (> 60% survivability), pH ranging from 2 to 12 (> 96% survivability), 0.5 to 15% (w/v) NaCl (> 98% survivability), chlorine up to 0.5% (v/v) (53% survivability), and chlorine neutralizer (100% survivability). In the animal challenge study, phage treatments, designed as “prevention” and “exclusion” programs, could control Salmonella on day 20 and 32 of the experiment, respectively; as indicated by the absence of Salmonella detection in cloacal swabs from broilers (0% prevalence). In the commercial-scale trial I, Salmonella was not detected in the phage-treated group from cloacal swabs, boot cover swabs, and bedding material samples after 16 days (0% prevalence) of phage administration. In the commercial-scale trial II, phage treatment extended the Salmonella control period in broilers during a 40-day growout period. In summary, a phage cocktail demonstrated high efficiency in controlling various serovars of Salmonella historically linked to contamination on these broiler farms. Phage cocktail application offers an effective, alternative to enhance food safety within the poultry value chain, protecting consumers and as well as the economic sustainability of the poultry sector.

Characterization of a Vibrio parahaemolyticus-targeting lytic bacteriophage SSJ01 and its application in artificial seawater.

Vibrio parahaemolyticus is a major foodborne pathogen causing serious illnesses. In this study, a new lytic bacteriophage SSJ01 that infects V. parahaemolyticus was isolated and characterized. It had a short non-contractile tail and belonged to the Caudoviricetes class. It rapidly adsorbed onto host cells, exhibited a short latent period, and has a large burst size. It showed lytic activities under a broad range of temperature (-18 to 60°C), pH (5 to 11), and salinity (0 to 6%). It contained 35 open reading frames with a G + C content of 49.16% without toxic or lysogen-forming genes. The MOI of 105 phage-treated group in vitro reduced the target cells up to 3.49-log CFU/mL at 6°C and 3.47-log CFU/mL at 25°C, respectively. In aquatic environments (6 and 25°C), bactericidal activities showed a significant decrease within 2h. Therefore, the bacteriophage SSJ01 has potential as a biocontrol agent to control V. parahaemolyticus in marine culture.

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.

Exploring the Feasibility of 16S rRNA Short Amplicon Sequencing-Based Microbiota Analysis for Microbiological Safety Assessment of Raw Oyster.

16S rRNA short amplicon sequencing-based microbiota profiling has been thought of and suggested as a feasible method to assess food safety. However, even if a comprehensive microbial information can be obtained by microbiota profiling, it would not be necessarily sufficient for all circumstances. To prove this, the feasibility of the most widely used V3-V4 amplicon sequencing method for food safety assessment was examined here. We designed a pathogen (Vibrio parahaemolyticus) contamination and/or V. parahaemolyticus-specific phage treatment model of raw oysters under improper storage temperature and monitored their microbial structure changes. The samples stored at refrigerator temperature (negative control, NC) and those that were stored at room temperature without any treatment (no treatment, NT) were included as control groups. The profiling results revealed that no statistical difference exists between the NT group and the pathogen spiked- and/or phage treated-groups even when the bacterial composition was compared at the possible lowest-rank taxa, family/genus level. In the beta-diversity analysis, all the samples except the NC group formed one distinct cluster. Notably, the samples with pathogen and/or phage addition did not form each cluster even though the enumerated number of V. parahaemolyticus in those samples were extremely different. These discrepant results indicate that the feasibility of 16S rRNA short amplicon sequencing should not be overgeneralized in microbiological safety assessment of food samples, such as raw oyster.

New findings about comparing the effects of antibiotic therapy and phage therapy on memory and hippocampal pyramidal cells in rats.

Pseudomonas aeruginosa is a significant cause of infection in burn wounds. Antibiotics are widely used to treat infectious diseases, and alongside their therapeutic benefits, they can damage host cells. Significant side effects, such as nephrotoxicity and neurotoxicity, are observed in 60% of patients treated with colistin. Therefore, using a suitable alternative instead of antibiotics is paramount. This study aimed to investigate the effects of phage therapy and antibiotic therapy on memory function in rats with P. aeruginosa infected burn wounds. Adult male rats were divided into three groups: (1) infected without treatment (control), (2) infected and treated with colistin antibiotic (3,000,000 international units/kg/day), and (3) infected and treated with 100 μL of phage suspension (approximately 109 PFU/mL). In all animals, after anesthesia, a third-degree burn was created in the back area. One hour later, treatment was performed for seven consecutive days. Passive avoidance test, novel object recognition test, locomotion activity, hippocampal neuron count, and oxidative stress measurement in blood serum were performed. In antibiotic-treated group memory recall, recognition index, number of healthy neurons in CA1, CA2, and CA3 hippocampus areas and the amounts of MDA, and FRAP significantly decreased compared with the control group. The phage-treated group was not shown any harmful effect on the memory process, number of healthy hippocampal neurons, and showed more positive effects in blood serum examinations compared with the antibiotic group. Phage therapy could be a safe and effective alternative to antibiotics in the treatment of burn-related infections.

Identification and characterization of the novel bacteriophage BPVP-3325 for the biocontrol of Vibrio parahaemolyticus infection in seafood

A total of 19 bacteriophages were isolated from seawater, wet sand, sea rocks, and suspended solids collected from 10 different sites in Busan, Republic of Korea. Among them, the best-performed novel phage was designated as BPVP-3325 that demonstrated the highest lytic activity in 76% of the total studied strains of Vibrio parahaemolyticus. The complete genome of the novel phage was 222,634 bp with 42% G + C content; electron microscopy showed that the total length and head length were 411.66 ± 6.69 and 129.87 ± 5.10 nm, respectively. In vitro characterization, V. parahaemolyticus ATCC 17802 was used as a standard pathogenic strain of BPVP-3325 host. The latent period was approximately 40 min, and the burst size was 6.76 PFU/infected cell. BPVP-3325 demonstrated excellent efficacy in a relatively wide pH range of up to 5–10 and temperature of <50 °C. When ATCC 17802 was treated with BPVP-3325 in a liquid medium for 6 h, OD600 in the phage-treated group was significantly lower (~50%) than that in the host. The application of BPVP-3325 to oyster meat effectively inhibited the growth and activity of the standard strain (P < 0.05). Therefore, this study described the potential of novel BPVP-3325 therapy for natural oyster culture and food industry for the biocontrol of V. parahaemolyticus disease in aquaculture and human food.

Isolation, characterization, and genome analysis of bacteriophage P929 that could specifically lyase the KL19 capsular type of Klebsiella pneumoniae

In recent years, Klebsiella pneumoniae has caused an increase in the number of serious infections associated with pneumonia, septicemia, urinary tract infections, and pyogenic liver abscess. In this study, a phage P929, isolated from hospital sewage in Jiangsu, could specifically infect K. pneumoniae KL19 capsular type by forming plaques with a translucent halo that expanded over time. Phage P929 with a multiplicity of infection (MOI) of 0.1 produced the highest phage titer. According to a one-step growth curve experiment, the latent time period of phage P929 was 25 min, and the burst size was about 156 phage particles/cell. The sensitivity tests confirmed that P929 was stable at temperatures ranging from 4 to 50 °C and pH 3 to 11. Based on morphological observation and phylogenetic analysis, phage P929 could be assigned to a new species in the genus Drulisvirus of the subfamily Slopekvirinae in the family Autographiviridae. According to genome analysis, phage P929 was 44,764 bp in size with 53.66% G + C content, encoding 57 proteins or coding sequences (117–3699 bp in length). Phage P929 showed potential antibacterial activity on planktonic cells and biofilm. After 120 min, the OD600 values of five phage-treated groups were basically reached zero compared to the untreated group, and the antibacterial activity of P929 was still detectable within 390 min. In anti-biofilm tests, phage P929 at an MOI of 1 significantly reduced the biofilm formation of K. pneumoniae in 48 h. These results suggest that phage P929 may be used to treat carbapenem-resistant and biofilm-forming K. pneumonia in clinical settings.

Phenotypic and Genetic Characterization of Aeromonas hydrophila Phage AhMtk13a and Evaluation of Its Therapeutic Potential on Simulated Aeromonas Infection in Danio rerio.

Phage therapy can be an effective alternative to standard antimicrobial chemotherapy for control of Aeromonas hydrophila infections in aquaculture. Aeromonas hydrophila-specific phages AhMtk13a and AhMtk13b were studied for basic biological properties and genome characteristics. Phage AhMtk13a (Myovirus, 163,879 bp genome, 41.21% CG content) was selected based on broad lytic spectrum and physiologic parameters indicating its lytic nature. The therapeutic potential of phage AhMtk13a was evaluated in experimental studies in zebrafish challenged with A. hydrophila GW3-10 via intraperitoneal injection and passive immersion in aquaria water. In experimental series 1 with single introduction of AhMtk13a phage to aquaria water at phage–bacteria ratio 10:1, cumulative mortality 44% and 62% was registered in fish exposed to phage immediately and in 4 h after bacterial challenge, correspondingly, compared to 78% mortality in the group with no added phage. In experimental series 2 with triple application of AhMtk13a phage at ratio 100:1, the mortality comprised 15% in phage-treated group compared to the 55% in the control group. Aeromonas hydrophila GW3-10 was not detectable in aquaria water from day 9 but still present in fish at low concentration. AhMtk13a phage was maintained in fish and water throughout the experiment at the higher concentration in infected fish.

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.

VB-ApyS-JF1, the First Trueperella pyogenes Phage, Shows Potential as an Alternative Treatment Strategy for Trueperella pyogenes Infections.

Trueperella pyogenes (T. pyogenes) is an important opportunistic animal pathogen that causes huge economic losses to the animal husbandry industry. The emergence of bacterial resistance and the unsatisfactory effect of the vaccine have prompted investigators to explore alternative strategies for controlling T. pyogenes infection. Due to the ability of phages to kill multidrug-resistant bacteria, the use of phage therapy to combat multidrug-resistant bacterial infections has attracted attention. In this study, a T. pyogenes phage, vB-ApyS-JF1 (JF1), was isolated from sewage samples, and its whole genome and biological characteristics were elucidated. Moreover, the protective effect of phage JF1 on a mouse bacteremic model caused by T. pyogenes was studied. JF1 harbors a double-stranded DNA genome with a length of 90,130 bp (30.57% G + C). The genome of JF1 lacked bacterial virulence–, antibiotic resistance– and lysogenesis-related genes. Moreover, the genome sequence of JF1 exhibited low coverage (<6%) with all published phages in the NCBI database, and a phylogenetic analysis of the terminase large subunits and capsid indicated that JF1 was evolutionarily distinct from known phages. In addition, JF1 was stable over a wide range of pH values (3 to 11) and temperatures (4 to 50°C) and exhibited strong lytic activity against T. pyogenes in vitro. In murine experiments, a single intraperitoneal administration of JF1 30 min post-inoculation provided 100% protection for mice against T. pyogenes infection. Compared to the phosphate-buffered saline (PBS) treatment group, JF1 significantly (P < 0.01) reduced the bacterial load in the blood and tissues of infected mice. Meanwhile, treatment with phage JF1 relieved the pathological symptoms observed in each tissue. Furthermore, the levels of the inflammatory cytokines tumour necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and interleukin-6 (IL-6) in the blood of infected mice were significantly (P < 0.01) decreased in the phage-treated group. Taken together, these results indicate that phage JF1 demonstrated great potential as an alternative therapeutic treatment against T. pyogenes infection.

Evaluation of the preventive effect of phage cocktails on turbot ascites and its influence on main physiological indicators

Edwardsiella tarda and Vibrio harveyi are the main pathogens causing ascites in turbot, which severely restricts the development of turbot farming. This study evaluated the effect of feeding phage cocktail on the prevention of ascites in turbot. The turbot was challenged by bacterial suspension comprising 50 μL of E. tarda ET9 at a concentration of 5.99 × 106 CFU/fish and 50 μL of V. harveyi VH5 at a concentration of 7.86 × 107 CFU/fish after they were fed with phage cocktails for 7 days. The cumulative survival rate of negative control group was 100%, and that of positive control group decreased to 7%, while that of phage-treated group was 60%, 83% and 87% when multiplicity of infection (MOI) was equal to 1, 10 and 100 respectively after 15 days of challenge. The pathological section showed that only the liver and intestines of turbot in the positive control group had lesions, while that of the phage-treated group had no obvious symptom. The contents of white blood cells, red blood cell distribution width and platelets in the blood of the positive control group were significantly higher than those of the negative control group and the phage-treated group (P < 0.05). In contrast, there was no significant difference between the negative control group and the phage-treated group (P > 0.05). Only the levels of Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Total protein (TP), Globulin (GLO), Total bile acid (TBA), Cholinesterase (CHE), Creatinine (CREA), Uric acid (UA), Glucose (GLU), Total cholesterol (CHOL), Low density lipoprotein (LDL), Creatine kinase (CK) and Creatine kinase isoenzymes (CKMB) in the serum of turbot in the positive control group were significantly different from those in the negative control group and the phage-treated group (P < 0.05), but there was no significant difference between the negative control group and the phage-treated group (P > 0.05). The transcription levels of 1L-1β, 1L-8, TNF-α and C 3 in the head kidney and spleen of turbot fed with phage cocktails with MOI = 10 were significantly lower than those of the positive control group after 24 h of challenge (P < 0.05). The intestinal flora of turbot in phage-treated group and positive control group was analyzed by high-throughput sequencing. The results showed that the abundance of Vibrio species and Edwardsiella species in turbot's intestine was significantly reduced by feeding with phage cocktails. These results indicate that feeding phage cocktails can effectively prevent bacterial ascites on turbot.

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 Efficacy of Phage Therapy in a Murine Model of Pseudomonas aeruginosa Pneumonia and Sepsis.

The emergence of multi-drug resistant Pseudomonas aeruginosa necessitates the search for treatment options other than antibiotic use. The use of bacteriophages is currently being considered as an alternative to antibiotics for the treatment of bacterial infections. A number of bacteriophages were introduced to treat pneumonia in past reports. However, there are still lack of knowledge regarding the dosages, application time, mechanism and safety of phage therapy against P. aeruginosa pneumonia. We used the bacteriophage KPP10 against P. aeruginosa strain D4-induced pneumonia mouse models and observed their outcomes in comparison to control models. We found that the nasal inhalation of highly concentrated KPP10 (MOI = 80) significantly improved survival rate in pneumonia models (P < 0.01). The number of viable bacteria in both lungs and in serum were significantly decreased (P < 0.01) in phage-treated mice in comparison to the control mice. Pathological examination showed that phage-treated group had significantly reduced bleeding, inflammatory cell infiltration, and mucus secretion in lung interstitium. We also measured inflammatory cytokine levels in the serum and lung homogenates of mice. In phage-treated models, serum TNFα, IL-1β, and IFN-γ levels were significantly lower (P < 0.05, P < 0.01, and P < 0.05, respectively) than those in the control models. In the lung homogenate, the mean IL-1β level in phage-treated models was significantly lower (P < 0.05) than that of the control group. We confirmed the presence of phage in blood and lungs, and evaluated the safety of bacteriophage use in living models since bacteriophage mediated bacterial lysis arise concern of endotoxic shock. The study results suggest that phage therapy can potentially be used in treating lung infections caused by Pseudomonas aeruginosa.

Locally isolated broad host-range bacteriophage kills methicillin-resistant Staphylococcus aureus in an in vivo skin excisional wound model in mice

From the perspective of an ever-increasing multidrug resistance among bacterial pathogens, bacteriophages are receiving renewed interest as potential alternative to antibiotics. We investigated the potential of a locally isolated species-specific phage against Staphylococcus aureus infection in a skin excisional wound model in mice. A significant time-dependent increase (P < 0.05) in wound healing was observed in the phage-treated mice groups. The animals treated with the phage ΦDMSA-2 exhibited a faster re-epithelialization and faster tissue re-modelling. Bacterial load in the infected tissue in all phage-treated groups diminished. The mean ± SD CFU per ml decreased from 3.3 × 108 ± 3.5 × 106 at day 1–1.43 × 103 ± 8.48 × 102 at day 16 (P < 0.05). The highest reduction in the bacterial load was observed in G5 (povidine-iodine treated) and G6 (povidine iodine + phage 107 PFU) groups as no bacterial counts were detected by day 12 in both groups. Interestingly, group G3, which was treated with a lower phage concentration (5 × 106 PFU), resulted in total clearing of the inoculated bacteria by day 16; while bacterial counts were still detected by that time in group G4, which was treated with a higher phage concentration of 107 PFU. Animals from phage-treated group G3 survived 100%, while those from the infected wound control group survived at a rate of 34% at day 9 and reached 0% by the end of day 22 (P < 0.001). The data from this study convincingly showed that phage treatment of the S. aureus-infected wounds resulted in a faster wound healing and a 100% survival of the animals. The results emphasize the utility of locally isolated species-specific phages in treatment against multidrug-resistant MRSA infections.

Characterization and genome analysis of Klebsiella phage P509, with lytic activity against clinical carbapenem-resistant Klebsiella pneumoniae of the KL64 capsular type.

The increasing population infected by carbapenem-resistant Klebsiella pneumoniae necessitates the development of alternative therapies. In this study, we isolated, characterized, and sequenced a bacteriophage, P509, which was able to specifically infect and lyse carbapenem-resistant K. pneumoniae of K locus type KL64. A one-step growth curve experiment showed that the latent time period of phage P509 was 5 min, and the burst size was about 85 phage particles/cell. Stability tests confirmed that P509 was stable over a wide range of temperatures (4 to 50°C) and pH (3 to 11) conditions. Phage P509 was identified as a linear double-stranded DNA phage with a genome of 40,954 bp with 53.2% G+C content, encoding 50 predicted proteins. Genomic and morphological analysis suggested that P509 belonged to the genusPrzondovirus, family Autographiviridae, order Caudovirales. Further analysis showed that no virulence-related genes or lysogen-formation gene clusters were detected in the genome, suggesting that P509 is a lytic phage, making it potentially suitable for clinical applications. In vitro, the number of viable cells in three phage-treated groups (MOI = 0.1, 0.01, 0.001) decreased by 3.75 log10 CFU/ml, 3.32 log10 CFU/ml and 3.21 log10 CFU/ml, respectively, after 80 min of incubation, in comparison to that in the untreated group. Based on these characteristics, phage P509 may be a promising candidate for future phage therapy applications.

Bacteriophage Protects Against Aerococcus viridans Infection in a Murine Mastitis Model.

Bovine mastitis, an inflammatory disease that occurs frequently in early lactation or the dry period, is primarily caused by bacterial infections. There is growing evidence that Aerococcus viridans (A. viridans) is becoming an important cause of bovine mastitis. The treatment of bovine mastitis is primarily based on antibiotics, which not only leads to a large economic burden but also the development of antibiotic resistance. On the other hand, bacteriophages present a promising alternative treatment strategy. The object of this study was to evaluate the potential of a previously isolated A. viridans phage vB_AviM_AVP (AVP) as an anti-mastitis agent in an experimental A. viridans-induced murine mastitis model. A. viridans N14 was isolated from the milk of clinical bovine mastitis and used to establish a mastitis model in mice. We demonstrated that administration of phage AVP significantly reduced colony formation by A. viridans and alleviated damage to breast tissue. In addition, reduced inflammation was indicated by decreased levels of inflammatory cytokines (TNF-α, IL-1β, and IL-6) and myeloperoxidase (MPO) activity in the phage-treated group compared to those in the phosphate buffered saline (PBS)-treated group. To the best of our knowledge, this report is the first to show the potential use of phages as a treatment for A. viridans-induced mastitis.

Proof-of-Principle Study in a Murine Lung Infection Model of Antipseudomonal Activity of Phage PEV20 in a Dry-Powder Formulation

Bacteriophage therapy is a promising alternative treatment to antibiotics, as it has been documented to be efficacious against multidrug-resistant bacteria with minimal side effects. Several groups have demonstrated the efficacy of phage suspension in vivo to treat lung infections using intranasal delivery; however, phage dry-powder administration to the lungs has not yet been explored. Powder formulations provide potential advantages over a liquid formulation, including easy storage, transport, and administration. The purpose of this study was to assess the bactericidal activities of phage dry-powder formulations against multidrug-resistant (MDR) strain Pseudomonas aeruginosa FADDI-PA001 in a mouse lung infection model. Phage PEV20 spray dried with lactose and leucine produced an inhalable powder at a concentration of 2 × 107 PFU/mg. P. aeruginosa lung infection was established by intratracheal administration of the bacterial suspension to neutropenic mice. At 2 h after the bacterial challenge, the infected mice were treated with 2 mg of the phage powder using a dry-powder insufflator. At 24 h after the phage treatment, the bacterial load in the lungs was decreased by 5.3 log10 (P < 0.0005) in the phage-treated group compared with that in the nontreated group. Additionally, the phage concentration in the lungs was increased by 1 log10 at 24 h in the treated group. These results demonstrate the feasibility of a pulmonary delivery of phage PEV20 dry-powder formulation for the treatment of lung infection caused by antibiotic-resistant P. aeruginosa.