Bacteriophages have applications in biotechnology, including human and veterinary medicine, agriculture, food safety, and biosecurity. One example of resurging importance is phage therapy, the use of phages to treat antibiotic-resistant bacterial infections. Phage therapy currently requires screening of environmental phages against the infecting strains for each individual patient, a laborious process that limits the development of standardized treatments. To overcome limitations of narrow host range inherent to many native phages, a robust genomic engineering platform is required which permits rapid and dependable genome production and engineering for nonmodel phage. Here, we describe an engineering platform for a phiKMV-like Pseudomonas aeruginosa phage, 41S1 that builds on previous work in the rapid assembly of small genomes through one-pot, High Complexity Golden Gate assembly (HC-GGA). This system divides the 41S1 genome into DNA fragments small enough to be conveniently synthesized and to avoid toxicity during DNA propagation, with all but one maintained in Escherichia coli. These fragments are readily assembled in a high accuracy, one-pot reaction; phages can be rescued by direct transformation into P. aeruginosa PAO1 or E. coli 10-beta cells. We demonstrate the precise generation of point mutations, DNA insertions, deletions, and the addition of fluorescence reporter genes that are expressed during phage replication. All viable genotypes could be generated with near 100% success rate with minimal screening. This system demonstrates the potential of HC-GGA for the rapid production and engineering of phages and provides a chassis for the development of phages that broadly target the opportunistic human pathogen P. aeruginosa.

Background Multidrug-resistant (MDR) pathogens such as Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae pose significant challenges to infection control and environmental sanitation, particularly in regions with high antimicrobial resistance (AMR) burdens. In 2021, Kenya reported approximately 28,500 deaths attributed to AMR, with K. pneumoniae accounting for the highest toll at 5220 deaths, followed by P. aeruginosa with 2,690, according to the Institute of Health Metrics. These pathogens are frequently detected in wastewater and hospital effluents, contributing to environmental persistence and transmission. Objective To isolate and descriptively characterise lytic bacteriophages (phages) targeting high-priority MDR bacteria from wastewater sources in Kilifi County, Kenya, with emphasis on genomic features that inform their safety, taxonomy, and potential relevance for future evaluation in applied contexts. Methods Four lytic phages were isolated using enrichment techniques against environmental MDR strains and purified by plaque assays. Whole-genome sequencing was performed using Oxford Nanopore technology, followed by annotation and comparative genomic analysis to determine taxonomic placement, conserved domains, and safety profiles. Results All four phages belong to the class Caudoviricetes and have genomic features consistent with obligately lytic replication, including the absence of lysogeny-associated genes and conserved structural modules. Comparative genomic analysis revealed global relatedness to previously described lytic phages. Preliminary spot-test observations demonstrated lytic activity against a limited set of local MDR isolates, providing initial descriptive evidence of their biological activity. Conclusion This study provides a genomics-based, preliminary characterisation of four wastewater-derived lytic phages. While their genomic profiles and observed activity suggest potential relevance for future evaluation in environmentally associated systems, further functional validation and application-orientated studies are required.

Decades of antibiotic overuse and misuse have placed us at the precipice of a post-antibiotic era. This rise in multidrug-resistant and pandrug-resistant bacteria poses a serious risk to public health, warranting urgent exploration of alternative treatment options for bacterial infections. One emerging method displaying promise in the field is phage-antibiotic combination therapy: the use of bacteriophages, viruses that exclusively infect bacteria, as adjuvants to antibiotics. This study investigated the synergistic effects of phage OMKO1, previously utilized in compassionate-use cases, with five antibiotics of diverse drug classes against Pseudomonas aeruginosa . Modified checkerboard assays were performed to test phage-antibiotic combination treatments across several antibiotic concentrations and phage multiplicities of infection. Synergy was achieved by four of the five phage-antibiotic pairings at sub-minimum inhibitory antibiotic concentrations. All combination treatments reduced the antibiotic minimum inhibitory concentration (MIC) by ≥ 2-fold and resulted in a reduction in resistant regrowth. These findings highlight the potential of phages to lower effective antibiotic concentrations and prolong their utility by slowing the rise of antimicrobial resistance. ### Competing Interest Statement The authors have declared no competing interest. Cystic Fibrosis Foundation, https://ror.org/00ax59295, 006478H223

Background: Pseudomonas aeruginosa, the most common sputum pathogen among adults with cystic fibrosis (CF), has become increasingly multidrug-resistant (MDR). To address this problem, we use lytic bacteriophages (phages) to design personalized phage therapy. Lipopolysaccharide (LPS) and Type IV Pilus (TIVP) are P. aeruginosa virulence factors that induce biofilm formation and lung inflammation. We investigate how LPS and TIVP targeting phage cocktail kills MDR P. aeruginosa and decreases biofilm formation and lung inflammation. Methods: Four patients with MDR P. aeruginosa were treated with LPS-5 and TIVP-H6 phage cocktail via FDA(eIND) and institutional review board approval. Phage was delivered via nebulization for 7 days. Sputum and spirometry were obtained before and after phage therapy. To evaluate biofilm formation, clinical isolates and a laboratory strain (PAO1) were cultured to mid-exponential growth phase (OD600=0.5). P. aeruginosa was co-cultured with a CF bronchial epithelial cell (CFBE41o-) line at an air-liquid interface or 6 hours. P. aeruginosa biofilms were collected and cultured overnight for CFU/ml comparison. To assess the phage therapy on CF inflammation, CFBE41o-cells were stimulated for 24 hours with P. aeruginosa cell-free supernatants. IL-6 was analyzed via sandwich ELISA. Results: After phage therapy sputum P. aeruginosa CFU/ml median decreased from 1.7 x108 to 5.2 x 104 a 4-log difference (p = 0.038). Precent predicted forced expiratory volume in 1 second improved from median 32% before therapy to 38% after therapy, a median difference of 6% (p = 0.086). Phage therapy decreased biofilm formation in day 15 isolates (N=3 isolates in duplicate, p = 0.0095). Phage therapy failed to decrease biofilm formation in day 27 isolates (N= 2 isolates in duplicate; p = 0.1429). Phage therapy reduced IL-6 secretion from CFBE41o-cells at 5 (p = 0.0062) and 15 days (p < 0.0001) after therapy. Conclusions: MDR P. aeruginosa is a common CF pathogen and exceedingly difficult to manage. Phage therapy is a novel way to investigate targeting bacteria virulence factors in the human lung.LPS-5 and TIVP-H6 phage cocktail therapy decreases P. aeruginosa biofilm formation and elicits an anti-inflammatory effect within 15 days after treatment. Deleterious effects on biofilm formation do not persist at day 27, suggesting a clinical window for phage retreatment. Investigations are ongoing into phage therapy effects on biofilm formation in the remaining patients, bacterial attachment, additional CF inflammatory markers, and CF animal models. This work contributes to developing improved personalized phage therapy against MDR P. aeruginosa in CF.

Background:Antimicrobial resistance is a major threat to human health worldwide, requiring investigation into alternative therapeutics such as phage therapy. Phages have been used to treat numerous multidrug-resistant infections recently, particularly in people with cystic fibrosis (CF) who are predisposed to infection from Pseudomonas aeruginosa. Case Summary:Here, we report the use of phage therapy to treat a chronic P. aeruginosa infection in a 44-year-old woman with CF who underwent bilateral lung transplantation. This individual experienced acute rejection and deteriorating lung function, possibly worsened as a consequence of a P. aeruginosa infection. Phage therapy using phage vB_Pae_10 was administered via nebulizer alongside standard antimicrobials in two 10-day courses. Clinical and microbiological assessments indicated an initial reduction in bacterial load along with an improvement in lung function. Phage therapy was well-tolerated with no adverse events reported. The patient showed sustained clinical improvement, including mucus clearance, enhanced pulmonary function, and resolution of acute rejection. Conclusion:This case underscores the potential of phage therapy to complement traditional treatments in managing chronic bacterial infections in CF patients, though further studies are needed to optimize treatment protocols and understand the role of phages in modulating the bacterial population.

Bacteriophage (phage) therapy, which uses lytic viruses as antimicrobials, is a potential strategy to address the antimicrobial resistance crisis. Cystic fibrosis, a disease complicated by recurrent Pseudomonas aeruginosa pulmonary infections, is an example of the clinical impact of antimicrobial resistance. Here, using a personalized phage therapy strategy that selects phages for a predicted evolutionary trade-off, nine adults with cystic fibrosis (eight women and one man) of median age 32 (range 22-46) years were treated with phages on a compassionate basis because their clinical course was complicated by multidrug-resistant or pan-drug-resistant Pseudomonas that was refractory to prior courses of standard antibiotics. The individuals received a nebulized cocktail or single-phage therapy without adverse events. Five to 18 days after phage therapy, sputum Pseudomonas decreased by a median of 104 CFU ml-1, or a mean difference of 102 CFU ml-1 (P = 0.006, two-way analysis of variance with Dunnett's multiple-comparisons test), without altering sputum microbiome, and an analysis of sputum Pseudomonas showed evidence of trade-offs that decreased antibiotic resistance or bacterial virulence. In addition, an improvement of 6% (median) and 8% (mean) predicted FEV1 was observed 21-35 days after phage therapy (P = 0.004, Wilcoxon signed-rank t-test), which may reflect the combined effects of decreased bacterial sputum density and phage-driven trade-offs. These results show that a personalized, nebulized phage therapy trade-off strategy may affect clinical and microbiologic endpoints, which must be evaluated in larger clinical trials.

Bacteriophage therapy is one potential strategy to treat antimicrobial resistant or persistent bacterial infections, and the year 2021 marked the centennial of Felix d’Hérelle’s first publication on the clinical applications of phages. At the Center for Phage Biology & Therapy at Yale University, a preparatory modular approach has been established to offer safe and potent phages for single-patient investigational new drug applications while recognizing the time constraints imposed by infection(s). This study provides a practical walkthrough of the pipeline with an Autographiviridae phage targeting Pseudomonas aeruginosa (phage vB_PaeA_SB, abbreviated to ΦSB). Notably, a thorough phage characterization and the evolutionary selection pressure exerted on bacteria by phages, analogous to antibiotics, are incorporated into the pipeline.

IntroductionAntimicrobial resistance in Latin America is a growing concern in both human and non-human animal populations. The economic burden that is likely to be imposed through increased resistance will cause further strains on public health systems and the population at large.Areas coveredWe propose the rapid adoption and implementation of phage therapy as a necessary addition to the medical arsenal to help mitigate antimicrobial resistance, with an emphasis on considering the potential benefits that highly biodiverse countries such as Ecuador may have on phage discovery. However, programs may count on limited government support and/or facilitation, which could slow progress.Expert opinionWe highlight the need for educational campaigns to be implemented in parallel with the development of phage therapy programs, particularly to implement these novel treatments in rural and indigenous communities.

BACKGROUND:Cystic fibrosis (CF) patients are prone to recurrent multi-drug-resistant (MDR) bacterial lung infections. Under this scenario, phage therapy has been proposed as a promising tool. However, the limited number of reported cases hampers the understanding of clinical outcomes. Anti-phage immune responses have often been overlooked and only described following invasive routes of administration. METHODS:Three monophage treatments against Staphylococcus aureus and/or Pseudomonas aeruginosa lung infections were conducted in cystic fibrosis patients. In-house phage preparations were nebulized over 10 days with standard-of-care antibiotics. Clinical indicators, bacterial counts, phage and antibiotic susceptibility, phage detection, and immune responses were monitored. FINDINGS:Bacterial load was reduced by 3-6 log in two of the treatments. No adverse events were described. Phages remained in sputum up to 33 days after completion of the treatment. In all cases, phage-neutralizing antibodies were detected in serum from 10 to 42 days post treatment, with this being the first report of anti-phage antibodies after nebulized therapy. CONCLUSIONS:Nebulized phage therapy reduced bacterial load, improving quality of life even without bacterial eradication. The emergence of antibodies emphasizes the importance of long-term monitoring to better understand clinical outcomes. These findings encourage the use of personalized monophage therapies in contrast to ready-to-use cocktails, which might induce undesirable antibody generation. FUNDING:This study was supported by the Spanish Ministry of Science, Innovation and Universities; Generalitat Valenciana; and a crowdfunding in collaboration with the Spanish Cystic Fibrosis Foundation.

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.

Cystic fibrosis (CF) is an important monogenic disease that affects more than 70 000 people worldwide. Defects of the CF transmembrane conductance regulator gene lead to dehydrated viscous secretions that result in chronic bacterial colonization. This leads to frequent recurrent lung infections called pulmonary exacerbations, lung inflammation, and resulting structural lung damage called bronchiectasis. Pseudomonas aeruginosa in particular is a common pathogen in persons with CF associated with increased pulmonary exacerbations, long-term lung function decline, and reduced survival. In addition, P. aeruginosa commonly develops antibiotic resistance and forms biofilms, making it difficult to treat. Here, we report the details of two patients with CF with pan-drug-resistant P. aeruginosa who were treated with a novel therapeutic strategy, bacteriophages. These cases highlight the need for further research and development of this treatment modality, including pediatric clinical trials.

Bacteriophage therapy is a promising treatment for periprosthetic joint infections (PJIs), particularly given these agents have innate abilities to degrade the biofilm matrix and lyse bacteria within. However, many aspects of this therapy are poorly understood causing treatments to lack uniform effectiveness and reproducibility, which is in part a consequence of several inherent limitations to using bacteriophages to treat PJI. Herein, these limitations are discussed as are additional translational research that needs to be conducted to advance this therapeutic. These include determining if bacteria causing PJIs are polyclonal, consequences of bacteriophage attachment receptor phenotypic variations and ramifications of bacteriophage activity when bacteria interact with in vivo macromolecules. Only with the realization of the current limitations and subsequent knowledge gained from translational research will the potential of bacteriophages to reduce the morbidity and mortality in PJI be fully elucidated.

Background:The antimicrobial resistance catastrophe is a growing global health threat and predicted to be worse in developing countries. Phages for Global Health (PGH) is training scientists in these regions to isolate relevant therapeutic phages for pathogenic bacteria within their locality, and thus contributing to making phage technology universally available.Materials and Methods:During the inaugural PGH workshop in East Africa, samples from Ugandan municipal sewage facilities were collected and two novel Escherichia coli lytic phages were isolated and characterized.Results:The phages, UP19 (capsid diameter ∼100 nm, contractile tail ∼120/20 nm) and UP30 (capsid diameter ∼70 nm, noncontractile tail of ∼170/20 nm), lysed ∼82% and ∼36% of the 11 clinical isolates examined, respectively. The genomes of UP19 (171.402 kb, 282 CDS) and UP30 (49.834 kb, 75 CDS) closely match the genera Dhakavirus and Tunavirus, respectively.Conclusion:The phages isolated have therapeutic potential for further development against E. coli infections.

We present the annotated genome sequence of Escherichia coli bacteriophage 107, a T4-like bacteriophage. Phage 107 has a genome length of 167,509 bp and 287 predicted genes.

We present the structural and functional annotation of Escherichia coli bacteriophage 55, which has a genome length of 170,393 bp, with 219 predicted genes.

Periprosthetic joint infections are a devastating complication of joint replacement surgery. One novel therapeutic that has potential to change the current treatment paradigm is bacteriophage therapy. Herein, we discuss our experiences with bacteriophage therapy for 10 recalcitrant periprosthetic joint infections and review the treatment protocols utilized to achieve successful outcomes.

Bacteriophage therapy has been suggested as an alternative or complementary strategy for the treatment of multidrug resistant (MDR) bacterial infections. Here, we report the favourable clinical evolution of a 41-year-old male patient with a Kartagener syndrome complicated by a life-threatening chronic MDR Pseudomonas aeruginosa infection, who is treated successfully with iterative aerosolized phage treatments specifically directed against the patient’s isolate. We follow the longitudinal evolution of both phage and bacterial loads during and after phage administration in respiratory samples. Phage titres in consecutive sputum samples indicate in patient phage replication. Phenotypic analysis and whole genome sequencing of sequential bacterial isolates reveals a clonal, but phenotypically diverse population of hypermutator strains. The MDR phenotype in the collected isolates is multifactorial and mainly due to spontaneous chromosomal mutations. All isolates recovered after phage treatment remain phage susceptible. These results demonstrate that clinically significant improvement is achievable by personalised phage therapy even in the absence of complete eradication of P. aeruginosa lung colonization.

Cystic fibrosis (CF) is an important monogenic disease that affects more than 70 000 people worldwide. Defects of the CF transmembrane conductance regulator gene lead to dehydrated viscous secretions that result in chronic bacterial colonization. This leads to frequent recurrent lung infections called pulmonary exacerbations, lung inflammation, and resulting structural lung damage called bronchiectasis. Pseudomonas aeruginosa in particular is a common pathogen in persons with CF associated with increased pulmonary exacerbations, long-term lung function decline, and reduced survival. In addition, P. aeruginosa commonly develops antibiotic resistance and forms biofilms, making it difficult to treat. Here, we report the details of two patients with CF with pan-drug-resistant P. aeruginosa who were treated with a novel therapeutic strategy, bacteriophages. These cases highlight the need for further research and development of this treatment modality, including pediatric clinical trials. Keywords cystic fibrosis , aeruginosa , drug resistance , microbial , bacteriophages , pediatrics

Chronic prosthetic joint infections are difficult to treat without conducting revision surgery because conventional antibiotics cannot eradicate bacteria that reside in biofilms. Consequently, novel therapeutics are needed to help treat prosthetic joint infections with one being bacteriophage therapy given its innate biofilm activity. Herein a sixty-nine-year-old man with a recalcitrant Enterococcus faecalis prosthetic joint infection is discussed. The patient was successfully treated with personalized bacteriophage therapy and after two years of follow up he has not had a clinical recurrence. Overall, this case report supports that bacteriophage therapy for prosthetic joint infections has promise to reduce the morbidity that is associated with current treatments. However, more research is needed to assess whether this therapeutic is helping eradicate infections or if it is making bacteria less pathogenic. This is an important point which will need to be evaluated as this therapeutic continues to be developed for all infections.