Phage Cocktail Research Articles

Isolation and characterisation of novel lytic bacteriophages for therapeutic applications in biofilm-associated prosthetic joint infections

Abstract Prosthetic joint infections are devastating complications of joint arthroplasties. Without effective management, they can lead to limb amputation and even death. A significant proportion of these infections is caused by the primarily commensal Coagulase-negative Staphylococci pathogens, which form thick, antibiotic-resistant biofilms at the site of infection. Combinatorial therapy involving antibiotics and bacteriophages may represent a strategy to overcome resistance. Previous research indicates that as bacteria develop resistance to antibiotics, they often become more susceptible to bacteriophages. In this study, we produced a cocktail of novel bacteriophages and assessed their viability to eradicate nosocomial staphylococcal biofilms. Here, we used clinical isolates from prosthetic joint infections to isolate and identify four new bacteriophages from sewage effluent. These novel phages were characterized through electron microscopy and full genome sequencing. Subsequently, we combined them into a phage cocktail, which effectively re-sensitized biofilms to vancomycin and flucloxacillin. Notably, this phage cocktail demonstrated low cytotoxicity in vitro to human epithelial cells, even when used alongside antibiotic treatments. These findings highlight the potential of the phage cocktail as a tool to increase antibiotic treatment success in prosthetic joint infections.

Synergistic effects of bacteriophage cocktail and antibiotics combinations against extensively drug-resistant Acinetobacter baumannii

BackgroundThe extensively drug-resistant (XDR) strains of Acinetobacter baumannii have become a major cause of nosocomial infections, increasing morbidity and mortality worldwide. Many different treatments, including phage therapy, are attractive ways to overcome the challenges of antibiotic resistance.MethodsThis study investigates the biofilm formation ability of 30 XDR A. baumannii isolates and the efficacy of a cocktail of four tempetate bacteriophages (SA1, Eve, Ftm, and Gln) and different antibiotics (ampicillin/sulbactam, meropenem, and colistin) in inhibiting and degrading the biofilms of these strains.ResultsThe majority (83.3%) of the strains exhibited strong biofilm formation. The bacteriophage cocktail showed varying degrees of effectiveness against A. baumannii biofilms, with higher concentrations generally leading to more significant inhibition and degradation rates. The antibiotics-bacteriophage cocktail combinations also enhanced the inhibition and degradation of biofilms.ConclusionThe findings suggested that the bacteriophage cocktail is an effective tool in combating A. baumannii biofilms, with its efficacy depending on the concentration. Combining antibiotics with the bacteriophage cocktail improved the inhibition and removal of biofilms, indicating a promising strategy for managing A. baumannii infections. These results contribute to our understanding of biofilm dynamics and the potential of bacteriophage cocktails as a novel therapeutic approach to combat antibiotic-resistant bacteria.

Making waves: Intelligent phage cocktail design, a pathway to precise microbial control in water systems

Current practices in water and wastewater treatment to control unwanted microbes have led to new problems, including health effects from disinfection byproducts, growth of opportunistic pathogens resistant to residual disinfectants (e.g., chlorine), and antibiotic resistance. These challenges are spurring interest in rethinking our practices of microbial control. Simultaneously, advances in molecular biology and computation power are driving renewed interest in using phages (viruses that infect bacteria) to precisely control microbial growth (aka, phage biocontrol). In this Making Waves article, I begin by reviewing the current state of research into phage cocktail design, emphasizing our limited understanding of the features of successful phage cocktails (combinations of multiple types of phages). I describe the state of modeling phage-bacteria interactions and underscore the need for increasing research efforts to predict phage cocktail success, a key gap slowing the application of phage biocontrol. I also detail how research must also focus on techniques for engineering more effective phages to offer a more rapid alternative to phage discovery from natural environments. In this way, phage cocktails comprised of phages with complementary infection strategies may be designed. The final area for increased research effort that I highlight is the need for phage cocktail design to account for possible unintended environmental effects, a risk that is increasingly acknowledged in phage ecology studies but mostly ignored by those developing phage biocontrol technologies. By focusing more research effort towards the areas necessary for intelligent phage cocktail design, we can accelerate the development of phage-based biocontrol in water systems and improve public health.

Bacteriophage-mediated approaches for biofilm control.

Biofilms are complex microbial communities in which planktonic and dormant bacteria are enveloped in extracellular polymeric substances (EPS) such as exopolysaccharides, proteins, lipids, and DNA. These multicellular structures present resistance to conventional antimicrobial treatments, including antibiotics. The formation of biofilms raises considerable concern in healthcare settings, biofilms can exacerbate infections in patients and compromise the integrity of medical devices employed during treatment. Similarly, certain bacterial species contribute to bulking, foaming, and biofilm development in water environments such as wastewater treatment plants, water reservoirs, and aquaculture facilities. Additionally, food production facilities provide ideal conditions for establishing bacterial biofilms, which can serve as reservoirs for foodborne pathogens. Efforts to combat antibiotic resistance involve exploring various strategies, including bacteriophage therapy. Research has been conducted on the effects of phages and their individual proteins to assess their potential for biofilm removal. However, challenges persist, prompting the examination of refined approaches such as drug-phage combination therapies, phage cocktails, and genetically modified phages for clinical applications. This review aims to highlight the progress regarding bacteriophage-based approaches for biofilm eradication in different settings.

Targeting Pseudomonas aeruginosa biofilm with an evolutionary trained bacteriophage cocktail exploiting phage resistance trade-offs

Spread of multidrug-resistant Pseudomonas aeruginosa strains threatens to render currently available antibiotics obsolete, with limited prospects for the development of new antibiotics. Lytic bacteriophages, the viruses of bacteria, represent a path to combat this threat. In vitro-directed evolution is traditionally applied to expand the bacteriophage host range or increase bacterial suppression in planktonic cultures. However, while up to 80% of human microbial infections are biofilm-associated, research towards targeted improvement of bacteriophages’ ability to combat biofilms remains scarce. This study aims at an in vitro biofilm evolution assay to improve multiple bacteriophage parameters in parallel and the optimisation of bacteriophage cocktail design by exploiting a bacterial bacteriophage resistance trade-off. The evolved bacteriophages show an expanded host spectrum, improved antimicrobial efficacy and enhanced antibiofilm performance, as assessed by isothermal microcalorimetry and quantitative polymerase chain reaction, respectively. Our two-phage cocktail reveals further improved antimicrobial efficacy without incurring dual-bacteriophage-resistance in treated bacteria. We anticipate this assay will allow a better understanding of phenotypic-genomic relationships in bacteriophages and enable the training of bacteriophages against other desired pathogens. This, in turn, will strengthen bacteriophage therapy as a treatment adjunct to improve clinical outcomes of multidrug-resistant bacterial infections.

Phage cocktail inhibits inflammation and protects the integrity of the intestinal barrier in dextran sulfate sodium-induced colitis mice model

Ulcerative colitis (UC) is an inflammatory bowel disease characterized by abdominal pain, diarrhea, and rectal bleeding. This study aims to explore the protective effects of a phage cocktail (108 PFU/mL of Clostridium perfringens phage, 108 PFU/mL of Escherichia coli phage, and 108 PFU/mL of Salmonella phage) on a mouse colitis model induced by dextran sulfate sodium (DSS) and its potential toxic effects on normal mice. The results demonstrate that the phage cocktail significantly alleviates clinical symptoms in mice, reduces colon shortening, weight loss, and colonic pathological damage. Furthermore, the phage cocktail markedly suppresses the inflammatory response and safeguards intestinal barrier integrity in the colonic tissues of the mouse colitis model. Preliminary investigation of the toxic effects of the phage cocktail in mice indicates that continuous administration for 14 days does not yield statistically significant differences in hematological and blood biochemical parameters, and specific pathological changes are absent in histopathological examination results. The aforementioned findings suggest that the phage cocktail exhibits anti-inflammatory and intestinal barrier protective effects in the mouse colitis model, and it does not exert significant toxic side effects on mice.

Isolation and evaluation of bacteriophage cocktail for the control of colistin-resistant Escherichia coli

The frequent emergence of colistin-resistant E. coli worldwide drives the exploration of alternative therapies, and bacteriophages (phages) have emerged as promising candidates to tackle this challenge. In this study, three E. coli phages were isolated, screened, and evaluated against 96 colistin-resistant strains obtained from diverse sources. The combined recognition rate for these strains was 43.6%, while individually it ranged from 17.0% to 24.5%. Notably, among the tested phages (FJ3-79, SD1-92L, and FJ4-63), FJ4-63 demonstrated exceptional characteristics in regulating host population dynamics upon infection by exhibiting a shorter latent period (20 min) and a larger burst size (95.99 ± 3.61 PFU/cell). Furthermore, it exhibited relative stability at pH 3-11 and below 60°C. Transmission electron microscopy and genomic analysis classified phage FJ4-63 belongs to the Dhakavirus genus within the Straboviridae family. Its genome comprised a linear double-stranded DNA measuring 169,669 bp (containing 272 coding sequences) with a GC content of 39.76%, of which 93 (34.2%) had known functions, and the remaining 177 were annotated as hypothetical proteins. Additionally, two tRNAs were recognized, possess the “holin-endolysin” lytic system, and no resistance or virulence genes were detected. The phylogenetic tree and average nucleotide identity (ANI) analysis revealed that phage FJ4-63 exhibited the highest similarity to Escherichia phage C6 (679410.1), indicating a consistent close relationship within the same branch. The cocktail comprising three phages exhibits enhanced in vitro bactericidal efficacy compared to a single phage. At high doses with MOI = 100, it rapidly and completely eradicates bacteria within 1 h while significantly reducing bacterial biofilms. All this evidence suggests that lytic phages offer an effective solution for clinical treatment, with a phage cocktail demonstrating greater potential in the alternative management of colistin-resistant E. coli infections.

Evaluating the Efficacy of Bacteriophage Therapy against Multidrug-Resistant Klebsiella pneumoniae Infections

Multidrug-resistant (MDR) Klebsiella pneumoniae poses a significant challenge to conventional antibiotic treatments. This study aims to assess the efficacy of bacteriophage therapy against MDR Klebsiella pneumoniae through a combination of in vitro assays, animal model experiments, and clinical case reviews. Phages isolated from environmental samples demonstrated potent lytic activity against MDR strains. In vitro assays showed that phages significantly reduced bacterial counts at a multiplicity of infection (MOI) of 1 or higher. Animal model experiments revealed that phage therapy, particularly using a cocktail of multiple phages, markedly improved survival rates and reduced bacterial loads in infected tissues compared to saline controls and standard antibiotics. Clinical case reviews indicated that patients receiving phage therapy experienced high rates of bacterial clearance and clinical improvement. These findings support the potential of bacteriophage therapy as an effective alternative or adjunct to traditional antibiotics in managing MDR Klebsiella pneumoniae infections.

A New Approach for Phage Cocktail Design in the Example of Anti-Mastitis Solution.

The studies on phage therapy have shown an overall protective effect of phages in bacterial infections, thus providing an optimistic outlook on the future benefits of phage-based technologies for treating bacterial diseases. However, the therapeutic effect is highly affected by the proper composition of phage cocktails. The rational approach to the design of bacteriophage cocktails, which is the subject of this study, allowed for development of an effective anti-mastitis solution, composed of virulent bacteriophages acting on Escherichia coli and Staphylococcus aureus. Based on the in-depth bioinformatic characterization of bacteriophages and their in vitro evaluation, the cocktail of five phages against E. coli and three against S. aureus strains was composed. Its testing in the milk model experiment revealed a reduction in the number of S. aureus of 45% and 30% for E. coli strains, and in the study of biofilm prevention, it demonstrated 99% inhibition of biofilm formation for all tested S. aureus strains and a minimum of 50% for 50% of E. coli strains. Such insights justify the need for rational design of cocktails for phage therapy and indicate the potential of the developed cocktail in the treatment of diseased animals, but this requires further investigations to evaluate its in vivo efficacy.

Genomic surveillance as a scalable framework for precision phage therapy against antibiotic-resistant pathogens

Phage therapy is gaining increasing interest in the fight against critically antibiotic-resistant nosocomial pathogens. However, the narrow host range of bacteriophages hampers the development of broadly effective phage therapeutics and demands precision approaches. Here, we combine large-scale phylogeographic analysis with high-throughput phage typing to guide the development of precision phage cocktails targeting carbapenem-resistant Acinetobacter baumannii, a top-priority pathogen. Our analysis reveals that a few strain types dominate infections in each world region, with their geographical distribution remaining stable within 6 years. As we demonstrate in Eastern Europe, this spatiotemporal distribution enables preemptive preparation of region-specific phage collections that target most local infections. Finally, we showcase the efficacy of phage cocktails against prevalent strain types using invitro and animal infection models. Ultimately, genomic surveillance identifies patients benefiting from the same phages across geographical scales, thus providing a scalable framework for precision phage therapy.

Ackermannviridae bacteriophage against carbapenem-resistant Klebsiella pneumoniae of capsular type 64.

Lytic bacteriophages (phages) are promising clinically viable therapeutic options against carbapenem-resistant Klebsiella pneumoniae (CRKP). In China, the predominant strains are those assigned to sequence type 11 and capsular type 64 (ST11-KL64). The emergence of phage resistance is a major bottleneck hindering effective phage therapy, requiring more new phages to provide the flexibility for creating different phage cocktails. However, the majority of phages against ST11-KL64 CRKP belong to the genus Przondovirus of the family Autographiviridae, which limits the options for constructing cocktails. We recovered a novel lytic phage of the genus Taipeivirus within the family Ackermannviridae against ST11-KL64 CRKP from a river in China. We phenotypically characterized this phage and obtained its genome sequence for analysis. This phage can inhibit the growth of ST11-KL64 CRKP for 6.5 h at a 0.1 multiplicity of infection and exhibits a narrow host range, being unable to attack CRKP strains of the other 30 capsular types. This phage carries no genes encoding antimicrobial resistance, virulence, or lysogeny. It is stable across a wide range of temperatures and pH values, making it suitable for phage therapy. Unlike other Taipeivirus phages, P01 has two tail spike proteins and a unique tail fiber protein. The distinct tail composition of this phage contributes to its activity against ST11-KL64 CRKP and its narrow host range. Taken together, we recovered a phage of a novel viral species with the potential for therapy, which expands the phage biobank against CRKP.

Assessment of the Safety of Anti-Salmonella Disinfectant for Veterinary Use Based on a Cocktail of Bacteriophages.

The toxicity and safety of a veterinary anti-salmonella disinfectant based on three highly virulent bacteriophage strains (titers 1010 PFU/ml) were studied. Acute, chronic, and inhalation toxicity, as well as local irritancy of the disinfectant were evaluated on outbred white mice CD1 (n=65), Soviet chinchilla rabbits (n=20), and rats (n=20). No toxic effects of the disinfectant was observed after its intraperitoneal or intragastric administration to mice and intragastric administration to rats; in rabbits, application on the skin and eyes produced no local irritation effect. Inhalation of 10% of the disinfectant did not cause any pathologies in mice. Thus, the tests confirmed the high level of safety of the disinfectant based on a mixture of bacteriophages for use as an additional specific disinfection agent against Salmonella in veterinary and livestock facilities.

Antibacterial effect of phage cocktails and phage-antibiotic synergy against pathogenic Klebsiella pneumoniae.

The worldwide spread of antimicrobial resistance (AMR) has posed a great challenge to global public health. Phage therapy has become a promising alternative against difficult-to-treat pathogens. One important goal of this study was to optimize the therapeutic efficiency of phage-antibiotic combinations, known as phage-antibiotic synergy (PAS). Through comprehensive analysis of the phenotypic and genotypic characteristics of a large number of CRKp-specific phages, we developed a systematic model for phage cocktail combinations. Crucially, our finding demonstrated that PAS treatments not only enhance the bactericidal effects of colistin and tigecycline against multidrug-resistant (MDR) K. pneumoniae strains in in vitro and in vivo context but also provide a robust response when antibiotics fail. Overall, the optimized PAS therapy demonstrates considerable potential in combating diverse K. pneumoniae pathogens, highlighting its relevance as a strategy to mitigate antibiotic resistance threats effectively.

PSVI-5 Bacteriophage biocontrol of avian pathogenic Escherichia coli in laying hens does not produce collateral effects on the cecal microbiota

Abstract Bacteriophages have shown promise as an alternative to antibiotics for managing or preventing avian pathogenic Escherichia coli (APEC) in the egg industry. In a previous study, a phage cocktail comprising 3 virulent phages was shown to prevent APEC infections in laying hens. The aim of this study was to investigate whether orally and intramuscularly administered phages affect the intestinal barrier function of chickens, with a focus on cecal microbiome. In a 4-d trial, a total of 35 laying hens were randomly assigned to one of four treatment groups: 1) Medium control group (bacterial broth + phage buffer), 2) APEC only control (APEC + buffer); 3) IM group (Phage +APEC), or 4) DW group (phage in drinking water (4 d prior to APEC + APEC). Cecum samples were subjected to 16S rRNA gene sequencing targeting the V4 region to assess the effects of phages on cecal bacterial diversity. A 1-way ANOVA was used to evaluate alpha diversity with respect to treatment and time. Microbial community structure was analyzed using β-dispersion and permutational multivariate analysis of variance (PERMANOVA) to determine the effect of each treatment. Significant (P < 0.05) differentially abundant genera were identified with DESeq2 by fitting a negative binomial model to each treatment vs treatment comparison (“~Treatment”). Across the treatment and control groups, 13 phyla were identified. Firmicutes (53.81%), Bacteroidota (23.83%), Actinobacteriota (1.84%), and Proteobacteria (1.02%) were the most abundant phyla among the amplicon sequence variants (ASVs), with 18.39% being unclassified. Neither richness (P = 0.448) nor Shannon diversity (P = 0.687) exhibited significance in α- diversity metric between treatment and control groups. β-dispersion analysis showed no significant difference (P = 0.239) between treatment and control groups, indicating similar bacterial variability among groups. Moreover, the β-diversity representing microbiota structure was not affected by any phage treatment (P = 0.083). In each treatment group, 76 unique genera were identified, with Megamonas (15.80 %), Lactobacillus (8.76%), Faecalibacterium (7.70 %), Megasphaera (1.58%), Alloprevotella (1.54%), [Ruminococcus] torques group (1.29%), Prevotellaceae UCG-001 (1.14%), Bifidobacterium (1.04%), and Olsenella (0.48%) being the most abundant. Notably, Lactobacillus was more abundant (~ 10 to 20%) in medium control group compared with phage treated and APEC only treatment groups. Furthermore, gene expression profiles of various genera (pairwise) were analyzed using log2 fold change (log2FC), revealing differences (P < 0.05) between control and treatment groups for a total of 58 genera. It was concluded that bacteriophage selectively killed APEC population without comprising the population and structure of cecal microbiome in laying hens.

Evaluation of lyophilized bacteriophage cocktail efficiency against multidrug-resistant Salmonella in broiler chickens

Currently, phage biocontrol is increasingly used as a green and natural technology for treating Salmonella and other infections, but phages exhibit instability and activity loss during storage. Therefore, in this study, the effects of lyophilization on the activity and stability of phage cocktails for the control of multidrug-resistant Salmonella in broiler chickens were determined. Eight serotypes of Salmonella were isolated and identified from broiler chicken farms, and bacteriophages against multidrug-resistant Salmonella enterica subsp. enterica serovar Kentucky, Salmonella enterica subsp. enterica serovar Typhimrium and Salmonella enterica subsp. enterica serovar Enteritidis were isolated. The bacteriophage cocktail was prepared and lyophilized, and it was subjected to in vitro and in vivo examinations. A reconstituted lyophilized bacteriophage cocktail was used for the oral treatment of chicks before and after challenge with multidrug-resistant S. Kentucky. The colonization of cecum by S. Kentucky was detected by using real-time PCR, and the serum levels of IgM, IgA and IL-4 and pathological changes in the different groups were detected. Three Caudovirales phages families were identified including Autographiviridae, Straboviridae and Drexlerviridae against multidrug-resistant S. Kentucky, S. Typhimrium and S. Enteritidis. The groups treated with the bacteriophage cocktail showed no clinical signs, no postmortem lesions, and a mortality rate of 0%, which improved the growth performance parameters. Additionally, the estimated serum levels of IgM, IgA and IL-4 were significantly greater in the bacteriophage cocktail-treated groups. Lyophilization effectively preserves the long-term storage stability of phages. Therefore, lyophilized bacteriophage cocktail therapy is a valuable approach for controlling multidrug-resistant Salmonella infections in broiler chickens.

Development and application of a bacteriophage cocktail for Shigella flexneri biofilm inhibition on the stainless steel surface

Food contamination and biofilm formation by Shigella in food processing facilities are major causes of acute gastrointestinal infection and mortality in humans. Bacteriophages (phages) are promising alternatives to antibiotics in controlling plankton and biofilms in food matrices. This study isolated two novel phages, S2_01 and S2_02, with lytic activity against various Shigella spp. From sewage samples. Transmission electron microscopy revealed that phages S2_01 and S2_02 belonged to the Caudovirales order. On characterizing their lytic ability, phage S2_01 initially exhibited relatively weak antibacterial activity, while phage S2_02 initially displayed rapid antibacterial activity after phage application. A combination of these phages in a 1:9 ratio was selected, as it has been suggested to elicit the most rapid and sustained lysis ability for up to 24 h. It demonstrated lytic activity against various foodborne pathogens, including six Shigella spp. The phage cocktail exhibited biofilm inhibition and disruption abilities of approximately 79.29% and 42.55%, respectively, after 24 h in a 96-well microplate. In addition, inhibition (up to 23.42%) and disruption (up to 19.89%) abilities were also observed on stainless steel surfaces, and plankton growth was also significantly suppressed. Therefore, the phage cocktail formulated in this study displays great potential as a biological control agent in improving food safety against biofilms and plankton.

Isolation, characterization, and potential application of Acinetobacter baumannii phages against extensively drug-resistant strains.

One of the significant issues in treating bacterial infections is the increasing prevalence of extensively drug-resistant (XDR) strains of Acinetobacter baumannii. In the face of limited or no viable treatment options for extensively drug-resistant (XDR) bacteria, there is a renewed interest in utilizing bacteriophages as a treatment option. Three Acinetobacter phages (vB_AbaS_Ftm, vB_AbaS_Eva, and vB_AbaS_Gln) were identified from hospital sewage and analyzed for their morphology, host ranges, and their genome sequences were determined and annotated. These phages and vB_AbaS_SA1 were combined to form a phage cocktail. The antibacterial effects of this cocktail and its combinations with selected antimicrobial agents were evaluated against the XDR A. baumannii strains. The phages exhibited siphovirus morphology. Out of a total of 30 XDR A. baumannii isolates, 33% were sensitive to vB_AbaS_Ftm, 30% to vB_AbaS_Gln, and 16.66% to vB_AbaS_Eva. When these phages were combined with antibiotics, they demonstrated a synergistic effect. The genome sizes of vB_AbaS_Ftm, vB_AbaS_Eva, and vB_AbaS_Gln were 48487, 50174, and 50043 base pairs (bp), respectively, and showed high similarity. Phage cocktail, when combined with antibiotics, showed synergistic effects on extensively drug-resistant (XDR) strains of A. baumannii. However, the need for further study to fully understand the mechanisms of action and potential limitations of using these phages is highlighted.

Multi-strain phage induced clearance of bacterial infections.

Bacteriophage (or 'phage' - viruses that infect and kill bacteria) are increasingly considered as a therapeutic alternative to treat antibiotic-resistant bacterial infections. However, bacteria can evolve resistance to phage, presenting a significant challenge to the near- and long-term success of phage therapeutics. Application of mixtures of multiple phage (i.e., 'cocktails') have been proposed to limit the emergence of phage-resistant bacterial mutants that could lead to therapeutic failure. Here, we combine theory and computational models of in vivo phage therapy to study the efficacy of a phage cocktail, composed of two complementary phages motivated by the example of Pseudomonas aeruginosa facing two phages that exploit different surface receptors, LUZ19v and PAK_P1. As confirmed in a Luria-Delbrück fluctuation test, this motivating example serves as a model for instances where bacteria are extremely unlikely to develop simultaneous resistance mutations against both phages. We then quantify therapeutic outcomes given single- or double-phage treatment models, as a function of phage traits and host immune strength. Building upon prior work showing monophage therapy efficacy in immunocompetent hosts, here we show that phage cocktails comprised of phage targeting independent bacterial receptors can improve treatment outcome in immunocompromised hosts and reduce the chance that pathogens simultaneously evolve resistance against phage combinations. The finding of phage cocktail efficacy is qualitatively robust to differences in virus-bacteria interactions and host immune dynamics. Altogether, the combined use of theory and computational analysis highlights the influence of viral life history traits and receptor complementarity when designing and deploying phage cocktails in immunocompetent and immunocompromised hosts.

Antibacterial efficacy of mycobacteriophages against virulent Mycobacterium tuberculosis

Tuberculosis (TB) remains a major global health concern, with drug-resistant strains posing a significant challenge to effective treatment. Bacteriophage (phage) therapy has emerged as a potential alternative to combat antibiotic resistance. In this study, we investigated the efficacy of widely used mycobacteriophages (D29, TM4, DS6A) against Mycobacterium tuberculosis (M. tuberculosis) under pathophysiological conditions associated with TB, such as low pH and hypoxia. We found that even at low multiplicity of infection (MOI), mycobacteriophages effectively infected M. tuberculosis, got rapidly amplified, and lysed M. tuberculosis, demonstrating their potential as therapeutic agents. Furthermore, we observed a novel phage tolerance mechanism with bacteria forming aggregates after several days of phage treatment. These aggregates were enriched with biofilm components and metabolically active bacteria. However, no phage tolerance was observed upon treatment with the three-phage mixture, highlighting the dynamic interplay between phages and bacteria and emphasizing the importance of phage cocktails. We also observed that phages were effective in lysing bacteria even under low pH and low oxygen concentrations as well as antibiotic-resistant bacteria. Our results provide key insights into phage infection of slow-growing bacteria and suggest that mycobacteriophages can effectively eliminate M. tuberculosis in complex pathophysiological environments like hypoxia and acidic pH. These results can aid in developing targeted phage-based therapies to combat antibiotic-resistant mycobacterial infections.

SalmoFree® Phage Additive Proves Its Safety for Laying Hens.

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