Phage Cocktail Research Articles

A Klebsiella-phage cocktail to broaden the host range and delay bacteriophage resistance both in vitro and in vivo.

Bacteriophages (phages), viruses capable of infecting and lysing bacteria, are a promising alternative for treating infections from hypervirulent, antibiotic-resistant pathogens like Klebsiella pneumoniae, though narrow host range and phage resistance remain challenges. In this study, the hypervirulent K. pneumoniae NTUH-K2044 was used to purify phage ΦK2044, while two ΦK2044-resistant strains were used to purify two further phages: ΦKR1, and ΦKR8 from hospital sewage. A detailed characterization showed that ΦK2044 specifically killed KL1 capsule-type K. pneumoniae, while ΦKR1 and ΦKR8 targeted 13 different capsular serotypes. The phage cocktail (ΦK2044 + ΦKR1 + ΦKR8) effectively killed K. pneumoniae in biofilms, pre-treatment biofilm formation, and delayed phage-resistance. The phage cocktail improved 7-day survival in Galleria mellonella and mouse models and showed therapeutic potential in a catheter biofilm model. In summary, this proof-of-principle phage cocktail has a broad host range, including hypervirulent and highly drug-resistant K. pneumoniae, and serves as a promising starting point for optimizing phage therapy.

Comprehensive Approaches to Combatting Acinetobacter baumannii Biofilms: From Biofilm Structure to Phage-Based Therapies

Acinetobacter baumannii—a multidrug-resistant (MDR) pathogen that causes, for example, skin and soft tissue wounds; urinary tract infections; pneumonia; bacteremia; and endocarditis, particularly due to its ability to form robust biofilms—poses a significant challenge in clinical settings. This structure protects the bacteria from immune responses and antibiotic treatments, making infections difficult to eradicate. Given the rise in antibiotic resistance, alternative therapeutic approaches are urgently needed. Bacteriophage-based strategies have emerged as a promising solution for combating A. baumannii biofilms. Phages, which are viruses that specifically infect bacteria, offer a targeted and effective means of disrupting biofilm and lysing bacterial cells. This review explores the current advancements in bacteriophage therapy, focusing on its potential for treating A. baumannii biofilm-related infections. We described the mechanisms by which phages interact with biofilms, the challenges in phage therapy implementation, and the strategies being developed to enhance its efficacy (phage cocktails, engineered phages, combination therapies with antibiotics). Understanding the role of bacteriophages in both biofilm disruption and in inhibition of its forming could pave the way for innovative treatments in combating MDR A. baumannii infections as well as the prevention of their development.

Leveraging mathematical modeling framework to guide regimen strategy for phage therapy

Bacteriophage (phage) cocktail therapy has been relied upon more and more to treat antibiotic-resistant infections. Understanding of the complex kinetics between phages, target bacteria, and the emergence of phage resistance remain hurdles to successful clinical outcomes. Building upon previous mathematical concepts, we develop biologically-motivated nonlinear ordinary differential equation models to explore single, cocktail, and sequential phage treatment modalities. While the optimal pairwise phage treatment strategy was the double simultaneous administration of two highly potent and asymmetrically binding phage strains, it appears unable to prevent the evolution of resistance. This treatment regimen did have a greater lysis efficiency, promoted higher phage population sizes, reduced bacterial density the most, and suppressed the evolution of resistance the longest compared to all other treatments strategies tested. Conversely, the combination of phages with polar potencies allows the more efficiently replicating phages to monopolize susceptible host cells, thereby quickly negating the intended compounding effect of cocktails. Together, we demonstrate that a biologically-motivated modeling-based framework can be leveraged to quantify the effects of each phage’s properties to more precisely predict treatment responses.

Refining the gut colonization Zophobas morio larvae model using an oral administration of multidrug-resistant Escherichia coli

The darkling beetle Zophobas morio can be implemented as an alternative in vivo model to study different intestinal colonization aspects. Recently, we showed that its larvae can be colonized by multidrug-resistant Escherichia coli strains administered via contaminated food (for 7 days) for a total experimental duration of 28 days. In the present work, we aimed to shorten the model to 14 days (T14) by administering the previously used CTX-M-15 ESBL-producing ST131 Escherichia coli strain Ec-4901.28 via a single oral administration (5 µL dose of 108 CFU/mL) , using a blunt 26s-gauge needle connected to a 250 μL gastight syringe. Force-feeding was performed either without or with (larvae placed on ice for 10 minutes before injection) anesthesia. In addition, phage-treated larvae were orally injected with 10 µL of INTESTI bacteriophage cocktail (∼105-6 PFU/mL) on days 4 (T4) and 7 (T7) . Growth curve analyses showed that, while larvae rapidly became colonized with Ec-4901.28 (T1, ∼106-7 CFU/mL) , only those anesthetized maintained a high bacterial load (∼102-3vs. ∼105-6 CFU/mL) and survival rate (76% vs. 99%; P<0.001) by T14. Moreover, bacteriophage administration to anesthetized larvae significantly reduced the bacterial count of INTESTI-susceptible Ec-4901.28 at T14 (5.17 × 105vs. 2.26 × 104, for non-treated and phage-treated larvae, respectively; P=0.04) . The methodological refinements applied to establish the intestinal colonization model simplify the use of Z. morio larvae, facilitate prompt evaluation of novel decolonization approaches and reduce experiments involving vertebrate animals in accordance with the Replacement, Reduction and Refinement principles.

Effect of novel phage cocktail on Salmonella recovered from broiler sources and its anti-biofilm effect on food contact surface model

The contamination of Salmonella in poultry products remains an important food safety and economic issue as the products have been recalled or rejected. Traditional antibiotic-based therapy in the animal production chain can cause antibiotic resistance (ABR) in bacteria, leading to severe illness and failure to treat diseases. Some strains of Salmonella produce genotoxin and extracellular polymeric substances called “biofilm” that support the survival of this bacterium. From this study, phages were applied to control cytolethal distending toxin B (CdtB)-producing, antibiotic-resistant, and biofilm-producing Salmonella isolated from the broiler sources. Out of 116 isolates, only 11 isolates (9.5%) representing six serovars were characterized. Of these, seven isolates were characterized as multidrug-resistant (MDR), three were resistant to one antibiotic, and only one isolate was susceptible to all antibiotics tested. For biofilm formation ability, 18.2% of the isolates were identified as strong biofilm producers, while 63.6% and 18.2% were moderate and weak, respectively. Up to 12 Salmonella phages from our collection were chosen to test their ability on given Salmonella. All the phages showed a strong lytic ability up to 90.9%. Three phages with the highest lysis potential (100% lytic ability) were further selected for a phage cocktail preparation, including WPX5, WPX8, and WPX9. Overall, the phage cocktail completely reduced representative Salmonella counts by three log units in vitro at a multiplicity of infection (MOI) of 104 and 105 at 6 h of treatment. The phage cocktail was tested to reduce Salmonella on different food contact surfaces. At MOI at 103, the highest reduction of Salmonella attachment was observed in stainless steel surfaces, indicated by a 67% reduction, followed by polyvinyl chloride (PVC) and ceramic by 64% and 58%, respectively. These results suggest that a developed phage cocktail could be a potential biocontrol to control toxin-producing, biofilm-producing, and MDR Salmonella in the poultry industry.

Relative fitness of wild-type and phage-resistant pyomelanogenic P. aeruginosa and effects of combinatorial therapy on resistant formation

Bacteriophages, the natural predators of bacteria, are incredibly potent candidates to counteract antimicrobial resistance (AMR). However, the rapid development of phage-resistant mutants challenges the potential of phage therapy. Understanding the mechanisms of bacterial adaptations to phage predation is crucial for phage-based prognostic applications. Phage cocktails and combinatorial therapy, using optimized dosage patterns of antibiotics, can negate the development of phage-resistant mutations and prolong therapeutic efficacy. In this study, we describe the characterization of a novel bacteriophage and the physiology of phage-resistant mutant developed during infection. M12PA is a P. aeruginosa-infecting bacteriophage with Myoviridae morphology. We observed that prolonged exposure of P. aeruginosa to M12PA resulted in the selection of phage-resistant mutants. Among the resistant mutants, pyomelanin-producing mutants, named PA-M, were developed at a frequency of 1 in 16. Compared to the wild-type, we show that PA-M mutant is severely defective in virulence properties, with altered motility, biofilm formation, growth rate, and antibiotic resistance profile. The PA-M mutant exhibited reduced pathogenesis in an allantoic-infected chick embryo model system compared to the wild-type. Finally, we provide evidence that combinatory therapy, combining M12PA with antibiotics or other phages, significantly delayed the emergence of resistant mutants. In conclusion, our study highlights the potential of combinatory phage therapy to delay the development of phage-resistant mutants and enhance the efficacy of phage-based treatments against P. aeruginosa.

Access to phage therapy at Hospices Civils de Lyon in 2022: Implementation of the PHAGEinLYON Clinic program

ObjectivesTo describe the set-up in 2022 of the PHAGEinLYON Clinic program dedicated to improve access to phage therapy in France using pharmaceutical-grade phages. MethodsWe described the process, prospectively collected all phage therapy requests received during 2022 and reviewed them retrospectively to analyze the decision and also the patient care pathway (NCT05883995). ResultsAmong 143 phage therapy requests, the indication was confirmed by multidisciplinary team meetings for 57 (40%) of them, and 44 were infected with bacteria that could be easily targeted by phages in France. Finally, 33 patients were treated, including 26 at our institution as compassionate use or in a clinical trial. Main indications were complex bone and joint infections, endovascular infections and lung infections. To manage these patients, 172 pharmaceutic phage cocktails targeting S. aureus and/or P. aeruginosa were prepared; 57 were dedicated to local, and 99 to intravenous injections. During the follow-up, 18 (69%) patients had a favorable clinical evolution, 6 (23%) required subsequent phage therapy, with the same phages with a greater phage exposition, or with different phages from elsewhere. ConclusionsThe implementation of the PHAGEinLYON Clinic program in 2022 was associated with a groundbreaking access of phage therapy in France.

Prediction of strain level phage-host interactions across the Escherichia genus using only genomic information.

Predicting bacteriophage infection of specific bacterial strains promises advancements in phage therapy and microbial ecology. Whether the dynamics of well-established phage-host model systems generalize to the wide diversity of microbes is currently unknown. Here we show that we could accurately predict the outcomes of phage-bacteria interactions at the strain level in natural isolates from the genus Escherichia using only genomic data (area under the receiver operating characteristic curve (AUROC) of 86%). We experimentally established a dataset of interactions between 403 diverse Escherichia strains and 96 phages. Most interactions are explained by adsorption factors as opposed to antiphage systems which play a marginal role. We trained predictive algorithms and pinpoint poorly predicted interactions to direct future research efforts. Finally, we established a pipeline to recommend tailored phage cocktails, demonstrating efficiency on 100 pathogenic E. coli isolates. This work provides quantitative insights into phage-host specificity and supports the use of predictive algorithms in phage therapy.

Directed evolution of bacteriophages: thwarted by prolific prophage.

Various directed evolution methods exist that seek to procure bacteriophages with expanded host ranges, typically targeting phage-resistant or non-permissive bacterial hosts. The general premise of these methods involves propagating phage(s) on multiple bacterial hosts, pooling the lysate, and repeating this process until phage(s) can form plaques on the target host(s). In theory, this produces a lysate containing input phages and their evolved phage progeny. However, in practice, this lysate can also include prophages originating from bacterial hosts. Here, we describe our experience implementing one directed evolution method, the Appelmans protocol, to study phage evolution in the Pseudomonas aeruginosa phage-host system, where we observed rapid host-range expansion of the phage cocktail. Further experimentation and sequencing revealed that the observed host-range expansion was due to a Casadabanvirus prophage originating from a lysogenic host that was only included in the first three rounds of the experiment. This prophage could infect five of eight bacterial hosts initially used, allowing it to persist and proliferate until the termination of the experiment. This prophage was represented in half of the sequenced phage samples isolated from the Appelmans experiment, but despite being subjected to directed evolution conditions, it does not appear to have evolved. This work highlights the impact of prophages in directed evolution experiments and the importance of genetically verifying output phages, particularly for those attempting to procure phages intended for phage therapy applications. This study also notes the usefulness of intraspecies antagonism assays between bacterial host strains to establish a baseline for inhibitory activity and determine the presence of prophage.IMPORTANCEDirected evolution is a common strategy for evolving phages to expand the host range, often targeting pathogenic strains of bacteria. In this study, we investigated phage host-range expansion using directed evolution in the Pseudomonas aeruginosa system. We show that prophages are active players in directed evolution and can contribute to observation of host-range expansion. Since prophages are prevalent in bacterial hosts, particularly pathogenic strains of bacteria, and all directed evolution approaches involve iteratively propagating phage on one or more bacterial hosts, the presence of prophage in phage preparations is a factor that needs to be considered in experimental design and interpretation of results. These results highlight the importance of screening for prophages either genetically or through intraspecies antagonism assays during selection of bacterial strains and will contribute to improving the experimental design of future directed evolution studies.

Computational prediction of a phage cocktail active against multidrug-resistant bacteria

Background Antibiotic misuse and overuse have contributed to the emergence of multi-drug resistant (MDR) bacteria, posing a serious public health problem across the globe. Phage cocktails, which combine multiple phages, provide an efficient method to combat multidrug-resistant bacterial infections. This study integrated a computational pipeline to design a phage cocktail against the bacterial strains Acinetobacter baumannii AB0057, Klebsiella pneumoniae subsp. pneumoniae HS11286, and Pseudomonas aeruginosa UCBPP-PA14. Methods The whole genome sequences of selected multidrug-resistant bacteria were accessed. Prophage sequences were identified from them which could be expressed to produce viable lytic phages against MDR bacterial strains, thereby reducing the severity of infection. Prophages were annotated for open reading frames (ORFs), putative promoters, virulence factors, transcriptional terminators, ribosomal RNAs, and transfer RNAs. A dot plot was also generated to investigate similar phages and phylogenetic analysis was performed. Results A total of 11 prophages were predicted from the bacterial genomes. About 472 open reading frames were predicted along with 3 transfer RNAs. Additionally, the presence of 754 putative promoters and 281 transcription terminator sequences was also detected. Comparative genomic and phylogenetic analyses provided insight into the diversity, relatedness, and lytic potential of the phages. The final designed phage cocktail consisted of five selected prophages including Acinetobacter baumannii prophages (2759376-2809756) and (3311844-3364667), and Klebsiella pneumoniae prophages (1288317-1338719), (1778306-1808606), and (2280703-2325555). Conclusion The phage cocktail designed in this study might be useful against MDR Acinetobacter baumannii and Klebsiella pneumoniae infections, especially where conventional antibiotics fail. Sequence similarity analysis suggested that the phage cocktail may also be effective against other carbapenemase-producing K. pneumoniae strains.

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.

Induced Native Phage Cocktails for Multi-microbial Activation Syndrome in Treatment-Resistant Illnesses.

The global population is plagued by chronic illnesses of many types due to, in part, the activation of virulence of many latent microbes leading to chronic multi-system illness, stimulating the new term, multi-microbial activation syndrome (MMAS). Treatment-resistant infections across the entire microbial spectrum are now largely untreatable using the previously successful pharmaceutical and natural medicine options. The heavy-handed methods of long-term chemotherapeutic antibiotics, antifungals, and antiviral drugs, whose adverse effects could be worse than the original illness, are presently essentially useless in the long run, as microbes have developed rapid adaptive mutations, becoming drug-resistant. The introduction of induced native phage therapy opened a completely new treatment category that has the potential to complement or replace aggressive drug therapies of many classes. Since its introduction, many advancements have been made to the technology. The most recent development in this new treatment genre is the ability to formulate induced native phage cocktails that use a wide spectrum of induction signatures within the formulation to stimulate various types of beneficial phages already living within the phageome of the body, to target a wide range of pathogenic microbes and associated advantageous physiological targets. The ability to address the MMAS commonly seen in various chronic illnesses simultaneously within the same formulation enables greater clinical outcomes without harm to the microbiome or to the tissues and systems of the body.

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.

Rapid design of bacteriophage cocktails to suppress the burden and virulence of gut-resident carbapenem-resistant Klebsiella pneumoniae

Antibiotic use can lead to the expansion of multi-drug-resistant pathobionts within the gut microbiome that can cause life-threatening infections. Selective alternatives to conventional antibiotics are in dire need. Here, we describe a Klebsiella PhageBank for the tailored design of bacteriophage cocktails to treat multi-drug-resistant Klebsiella pneumoniae. Using a transposon library in carbapenem-resistant K.pneumoniae, we identify host factors required for phage infection in major Klebsiella phage families. Leveraging the diversity of the PhageBank, we formulate phage combinations that eliminate K.pneumoniae with minimal phage resistance. Optimized cocktails selectively suppress the burden of K.pneumoniae in the mouse gut and drive the loss of key virulence factors that act as phage receptors. Phage-mediated diversification of bacterial populations in the gut leads to co-evolution of phage variants with higher virulence and broader host range. Altogether, the Klebsiella PhageBank charts a roadmap for phage therapy against a critical multidrug-resistant human pathogen.

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.