The rise of antibiotic-resistant infections, particularly those involving biofilms, presents a significant global health threat. Phage therapy, the use of bacteriophages as antimicrobial agents, offers promising solutions to this crisis. A critical component of phage therapy is the assessment of phage efficacy, in both the presence and absence of antibiotics, prior to clinical application. While considerable progress has been made using planktonic bacterial cultures, there remains an urgent need for standardized methods to evaluate phage efficacy against biofilms. In this study, we address this gap by systematically comparing ten different methods for quantifying phage activity in biofilm settings. Each method was evaluated using a panel of five anti-Pseudomonas aeruginosa phages, which were tested against both planktonic and biofilm cultures. Based on these comparisons, we propose a robust pipeline for detecting phage activity in biofilms. This pipeline, termed CApEsid biOfilm, integrates modified colony-forming unit (CFU) assays using stainless steel washers, crystal violet staining, extracellular DNA quantification using a dye, and extracellular ATP measurements. The pipeline was further validated with additional bacterial species and their respective phages. We also demonstrate its utility in detecting interactions between phages and antibiotics. Overall, this work presents a foundational pipeline that may enhance the clinical matching of phages for treating biofilm-associated infections, thereby improving the outcomes of phage therapy.

Background: Bariatric surgery (BaS) is a safe and effective treatment for severe obesity, yet recent studies suggested that it may impact the gut and oral microbiomes. Oral dysbiosis is associated with an increased risk of oral diseases, including periodontitis and dental caries. However, the effects of BaS on oral health and microbiome changes are poorly understood. Objectives: To examine the impact of obesity and BaS on oral health and microbiota in obese patients and an induced experimental periodontitis (EP) obese mouse model. Methods: The oral health of pre-BaS, post-BaS (6 months post-surgery), and control volunteers was assessed by scoring gingivitis, caries, and periodontitis. The oral microbiome was analyzed using 16S rRNA Next-Generation sequencing (NGS). Systemic parameters, maxillary bone volume, and oral and fecal microbiome were investigated in male obese C57BL/6 mice with and without EP before and after BaS treatment. Results: The study included 36 pre-BaS patients, 14 post-BaS patients, and 56 controls. Distinct oral microbial profiles were noted for each cohort. Pre-BaS patients exhibited higher oral microbial diversity and a greater prevalence of periodontitis-associated bacteria than controls, which further increased post-BaS. Caries- and halitosis-associated bacteria were significantly more abundant after BaS. Similarly, obese mice with EP and that underwent BaS showed elevated oral microbial diversity, paralleling human findings. EP alone significantly reduced gut microbiome diversity, regardless of BaS. Conclusions: BaS exacerbates obesity-related microbial dysbiosis, increasing the risk of periodontal and dental diseases. Paradoxically, while BaS improves systemic and cardiometabolic health, it appears to worsen oral outcomes, underscoring the need for integrated medical–dental care and preventive protocols in this population.

Introduction: Pseudomonas aeruginosa is an opportunistic pathogen that causes health care-associated infections. The rise of antibiotic-resistant bacterial strains necessitates alternative treatment strategies, with bacteriophage therapy being a promising approach.Methods: Six bacteriophages were isolated from sewage samples. Phage isolation involved centrifugation, filtration, and plaque assays. The morphology of each sample was examined using transmission electron microscopy (TEM). Genomic DNA was sequenced and compared among the isolates. The phages' lytic activities were assessed using growth curve analysis.Results: The phages displayed distinct genomic characteristics, grouping into three genomic clusters. No known virulence or antibiotic resistance genes were detected, indicating their safety for therapeutic use. Taxonomic analysis identified the phages as belonging to the genera Pbunavirus, Nipunavirus, Abidjanvirus, and a novel genus. TEM analysis revealed their diverse morphologies. Temperate phages showed less effective lytic activities.Conclusion: Several of the isolated bacteriophages show potential as candidates for phage therapy research and could be effective against P. aeruginosa infections.

Abstract Non-resolving bacterial infections involve biofilm formation and are often complicating treatments. Utilizing lytic bacteriophages with antibiotics holds promise in biofilm eradication. Accurately matching phage-antibiotic combinations against target bacteria, termed Clinical Phage Microbiology (CPM), is crucial in effective phage therapy treatments. However, compared to planktonic cultures, performing CPM on biofilm infections poses a significant challenge due to the lack of effective methods and a standard protocol. This study compared various CPM approaches in biofilms. To this end, the activity of five phages was assessed against Pseudomonas aeruginosa biofilms using nine methods and multiple approaches. Here, we discuss various aspects of each technique, including sensitivity, duration, ease of implementation in diagnostic labs, and labor. Finally, we offer a preliminary protocol for testing phage-sensitivity in biofilm, which was tested on various bacteria species, and may serve as a basis for comprehensive CPM in biofilm. ### Competing Interest Statement The authors have declared no competing interest.

Background: Antibiotic-resistant Pseudomonas aeruginosa (P. aeruginosa) strains are an increasing cause of morbidity and mortality. Pulsed blue light (PBL) enhances porphyrin-induced reactive oxygen species and has been clinically shown to be harmless to the skin at low doses. Bacteriophages, viruses that infect bacteria, offer a promising non-antibiotic bactericidal approach. This study investigates the potential synergism between low-dose PBL and phage therapy against P. aeruginosa in planktonic cultures and preformed biofilms. Methods: We conducted a factorial dose–response in vitro study combining P. aeruginosa-specific phages with PBL (457 nm, 33 kHz) on both PA14 and multidrug-resistant PATZ2 strains. After excluding direct PBL effects on phage titer or activity, we assessed effectiveness on planktonic cultures using growth curve analysis (via growth_curve_outcomes, a newly developed, Python-based tool available on GitHub) , CFU, and PFU. Biofilm efficacy was evaluated using CFU post-sonication, crystal violet staining, and live/dead staining with confocal microscopy. Finally, we assessed reactive oxygen species (ROS) as a potential mechanism using the nitro blue tetrazolium reduction assay. ANOVA or Kruskal–Wallis tests with post hoc Tukey or Conover–Iman tests were used for comparisons (n = 5 biological replicates and technical triplicates). Results: The bacterial growth lag phase was significantly extended for phage alone or PBL alone, with a synergistic effect of up to 144% (p < 0.001 for all), achieving a 9 log CFU/mL reduction at 24 h (p < 0.001). In preformed biofilms, synergistic combinations significantly reduced biofilm biomass and bacterial viability (% Live, median (IQR): Control 80%; Phage 40%; PBL 25%; PBL&Phage 15%, p < 0.001). Mechanistically, PBL triggered transient ROS in planktonic cultures, amplified by phage co-treatment, while a biphasic ROS pattern in biofilms reflected time-dependent synergy. Conclusions: Phage therapy combined with PBL demonstrates a synergistic bactericidal effect against P. aeruginosa in both planktonic cultures and biofilms. Given the strong safety profile of PBL and phages, this approach may lead to a novel, antibiotic-complementary, safe treatment modality for patients suffering from difficult-to-treat antibiotic-resistant infections and biofilm-associated infections.

Background Systemic alterations in the oral cavity can be reflected in skin disorders like psoriasis. However, data about oral health factors that are affected and controlled mainly by oral microbiota in atopic dermatitis (AD) are sparse. This study compared the oral status and oral microbiota of AD patients and healthy controls. Methods This was a prospective sex- and age-matched case-control study comparing adult participants with and without dermatologist-verified AD. A dentist assessed oral health status, and oral flora samples were collected and subjected to 16S rRNA sequencing for microbiome analysis. Results Forty-five AD participants and 41 non-AD controls were recruited. Compared to the participants in the control group, those with AD had significantly higher plaque levels (P = .04), poorer oral hygiene indices (P = .04), and higher gingival index trends (P = .05). The oral microbiome in the AD group showed significantly higher diversity, both in α and β diversities (P = .001, P = .0007, respectively). Furthermore, AD patients had a significantly increased abundance of taxa correlated with oral diseases and a decreased abundance of bacteria associated with a healthy oral status. Conclusion AD appears to be associated with poor oral health and oral dysbiosis. There is a need to increase both patients’ and physicians’ awareness of oral health.

Antibiotic-resistant pathogens are a growing global issue, leading to untreatable infectious diseases in both humans and animals. Personalized bacteriophage (phage) therapy, the use of specific anti-bacterial viruses, is currently a leading approach to combat antibiotic-resistant infections. The implementation of phage therapy has primarily been focused on humans, almost neglecting the impact of such infections on the health and welfare of companion animals. Pets also have the potential to spread resistant infections to their owners or the veterinary staff through zoonotic transmission. Here, we showcase personalized phage-antibiotic treatment of a cat with a multidrug-resistant Pseudomonas aeruginosa implant-associated infection post-arthrodesis surgery. The treatment encompassed a tailored combination of an anti-P. aeruginosa phage and ceftazidime, precisely matched to the pathogen. The phage was topically applied to the surgical wound while the antibiotic was administered intramuscularly. After two treatment courses spanning 7 and 3 weeks, the surgical wound, which had previously remained open for five months, fully closed. To the best of our knowledge, this is the first case of personalized phage therapy application in felines, which provides further evidence of the effectiveness of this approach. The successful outcome paves the way for personalized phage-antibiotic treatments against persistent infections therapy in veterinary practice.

Phage therapy has re-emerged as a promising treatment for non -resolving infections. Given the lack of approved phage treatments, there is a need to establish a compassionate use pipeline. Here, we present a protocol for phage matching, treatment, and monitoring for compassionate bacteriophage use in nonresolving infections. We describe steps for consultation and request implementation, evaluating and comparing different aspects of phage activity, and phage production. We then detail procedures for multidisciplinary meetings, ethics approvals, phage therapy, and follow-up. For complete details on the use and execution of this protocol, please refer to Onallah et al. 1,2

Antibiotic-resistant Pseudomonas aeruginosa (PA) is a critical health threat. Novel treatment approaches are urgently required in this post-antibiotic era. In the current study, we investigated the bactericidal combinatorial potential of two non-antibiotic alternative approaches: phage therapy and pulsed blue light (PBL). Bacteriophages (phages), are viruses that specifically infect and lyse bacteria without harming eukaryotic cells. Pulsed blue light (PBL) alters bacterial membranes and was clinically shown to be innocuous to the skin in low doses. Here, using a low dose 457nm, 33KHz PBL combined with specific PA targeting phages, we demonstrated a synergistic effect that achieved complete inhibition of planktonic bacteria and a 40% reduction in formed biofilms. As part of this study, we also developed a user-friendly python-based tool for extraction of growth curve outcomes. In vivo studies are warranted for further validation of this combinatorial treatment. This approach may lead to a novel, antibiotic complementary modality to help patients suffering from difficult-to-treat antibiotic-resistant infections. ### Competing Interest Statement The authors have declared no competing interest.

PurposeBurkholderia cepacia complex (BCC) are uncommon pathogens but associated with high morbidity/ mortality. Antibiotics are limited and phage therapy is being explored as a potential alternative. Our goal was to develop a phage library to target clinical BCC isolates for use in compassionate use protocols and clinical trials.MethodsWe developed an international, multicenter registry in which BCC-infected persons with CF were identified and entered into a coded dataset. Clinical isolates from patients (US, Canada) were sent to a central repository (JL, where bacteria were genotyped and stored) and then sent to a research laboratory (RH, where phage hunt was performed). Traditional methods of phage discovery were utilized to identify lytic phages. Phage characterization included determination of lytic activity, whole-genome sequencing, and host range determination. In addition to registry patients, additional BCC laboratory isolates were collected to assess phage coverage.ResultsFrom 7/1/2020- 9/30/2022, 56 patients were enrolled with baseline details noted in Table 1. BCC isolates consisted of B. cepacia (4), B. cenocepacia (14), B. multivorans (14), B. vietnamiensis (3), B. dolosa (3), B. gladioli (14), B. stabilis (1) and other (3). Additional 44 BCC laboratory isolates were added for total 100 BCC isolates. We identified 19 unique phages that were active against 80% of 35 tested isolates (work is ongoing) as noted in Table 2. Among these, 3 phages were active against 50% of the isolates. Among 6 patients that initiated compassionate use evaluation, phage(s) were found for 5. One patient was treated with phage prior to registry entry, two are awaiting clinical use, and two expired prior to phage use. Genetic sequencing of phages and phage-antibiotic synergy testing is ongoing.ConclusionWe developed a multicenter registry of BCC-infected patients and used clinical isolates to develop a targeted lytic phage library of 19 phages that covered 80% of isolates. Burkholderia cepacia complex (BCC) are uncommon pathogens but associated with high morbidity/ mortality. Antibiotics are limited and phage therapy is being explored as a potential alternative. Our goal was to develop a phage library to target clinical BCC isolates for use in compassionate use protocols and clinical trials. We developed an international, multicenter registry in which BCC-infected persons with CF were identified and entered into a coded dataset. Clinical isolates from patients (US, Canada) were sent to a central repository (JL, where bacteria were genotyped and stored) and then sent to a research laboratory (RH, where phage hunt was performed). Traditional methods of phage discovery were utilized to identify lytic phages. Phage characterization included determination of lytic activity, whole-genome sequencing, and host range determination. In addition to registry patients, additional BCC laboratory isolates were collected to assess phage coverage. From 7/1/2020- 9/30/2022, 56 patients were enrolled with baseline details noted in Table 1. BCC isolates consisted of B. cepacia (4), B. cenocepacia (14), B. multivorans (14), B. vietnamiensis (3), B. dolosa (3), B. gladioli (14), B. stabilis (1) and other (3). Additional 44 BCC laboratory isolates were added for total 100 BCC isolates. We identified 19 unique phages that were active against 80% of 35 tested isolates (work is ongoing) as noted in Table 2. Among these, 3 phages were active against 50% of the isolates. Among 6 patients that initiated compassionate use evaluation, phage(s) were found for 5. One patient was treated with phage prior to registry entry, two are awaiting clinical use, and two expired prior to phage use. Genetic sequencing of phages and phage-antibiotic synergy testing is ongoing. We developed a multicenter registry of BCC-infected patients and used clinical isolates to develop a targeted lytic phage library of 19 phages that covered 80% of isolates.

Acne vulgaris is a common neutrophil-driven inflammatory skin disorder in which Cutibacterium acnes (C. acnes) is known to play a key role. For decades, antibiotics have been widely employed to treat acne vulgaris, inevitably resulting in increased bacterial antibiotic resistance. Phage therapy is a promising strategy to combat the growing challenge of antibiotic-resistant bacteria, utilizing viruses that specifically lyse bacteria. Herein, we explore the feasibility of phage therapy against C. acnes. Eight novel phages, isolated in our laboratory, and commonly used antibiotics eradicate 100% of clinically isolated C. acnes strains. Topical phage therapy in a C. acnes-induced acne-like lesions mouse model affords significantly superior clinical and histological scores. Moreover, the decrease in inflammatory response was reflected by the reduced expression of chemokine CXCL2, neutrophil infiltration, and other inflammatory cytokines when compared with the infected-untreated group. Overall, these findings indicate the potential of phage therapy for acne vulgaris as an additional tool to conventional antibiotics.

Background: A growing number of compassionate phage therapy cases were reported in the last decade, with a limited number of clinical trials conducted and few unsuccessful clinical trials reported. There is only a little evidence on the role of phages in refractory infections. Our objective here was to present the largest compassionate-use single-organism/phage case series in 16 patients with non-resolving Pseudomonas aeruginosa infections. Methods: We summarized clinical phage microbiology susceptibility data, administration protocol, clinical data, and outcomes of all cases treated with PASA16 phage. In all intravenous phage administrations, PASA16 phage was manufactured and provided pro bono by Adaptive Phage Therapeutics. PASA16 was administered intravenously, locally to infection site, or by topical use to 16 patients, with data available for 15 patients, mainly with osteoarticular and foreign-device-associated infections. Findings: A few minor side effects were noted, including elevated liver function enzymes and a transient reduction in white blood cell count. Good clinical outcome was documented in 13 out of 15 patients (86.6%). Two clinical failures were reported. The minimum therapy duration was 8 days with a once-to twice-daily regimen. Conclusions: PASA16 with antibiotics was found to be relatively successful in patients for whom traditional treatment approaches have failed previously. Such pre-phase-1 cohorts can outline potential clinical protocols and facilitate the design of future trials.

SUMMARYAcne vulgaris is a common neutrophile-driven inflammatory skin disorder in whichCutibacterium acnes(C. acnes) bacteria play a significant role. Until now, antibiotics have been widely used to treat acne vulgaris, with the inevitable increase in bacterial antibiotic resistance. Phage therapy is a promising solution to the rising problem of antibiotic-resistant bacteria, utilizing viruses that specifically lyse bacteria.Here, we explored the feasibility of phage therapy againstC. acnes. By combining eight novel phages we had isolated, together with commonly used antibiotics, we achieved 100% eradication of clinically isolatedC. acnesstrains. Using topical phage therapy in an acne mouse model resulted in significantly superior clinical scores, as well as a reduction in neutrophil infiltration compared to the control group. These results demonstrate the potential of phage therapy in acne vulgaris treatment, especially when antibiotic-resistant strains are involved.

Abstract Acne vulgaris is a common neutrophil-driven inflammatory skin disorder in which Cutibacterium acnes (C. acnes) plays a significant role. For decades antibiotics have been widely used to treat acne vulgaris, with the inevitable increase in bacterial antibiotic resistance.Phage therapy is a promising solution to the rising problem of antibiotic-resistant bacteria, utilizing viruses that specifically lyse bacteria.Here, we explored the feasibility of phage therapy against C. acnes. Combining eight novel phages we had isolated and commonly used antibiotics, 100% of clinically isolated C. acnes strains were eradicated.Using topical phage therapy in a C. acnes-induced acne-like lesions mouse model resulted in significantly superior clinical and histological scores. Moreover, the inflammatory response decreased as reflected by reducing the chemokine CXCL2, infiltration of neutrophils, and other inflammatory cytokines, compared to the untreated group.These results demonstrate the potential of phage therapy in acne vulgaris treatment, especially when antibiotic-resistant strains are involved.

PASA16 is a Pseudomonas aeruginosa phage isolated from a soil sample and used to treat several patients suffering from persistent infections in various countries. PASA16’s genome was sequenced, analyzed, and deposited in GenBank.

The fascinating scientific history of phage therapy has been documented in numerous publications. In this study, however, we focus on an angle of the story that hitherto has remained relatively neglected, namely, phage therapy treatments, and the protagonists that conducted these in Mandatory-Palestine and subsequently the state of Israel, as part of a global trend. We complete the story by describing efforts in the new era of phage therapy in present-day Israel.

Providencia spp. are emerging pathogens mainly in nosocomial infections. Providencia stuartii in particular is involved in urinary tract infections and contributes significantly to the high incidence of biofilm-formation in catheterized patients. Furthermore, recent reports suggested a role for multiple drug resistant (MDR) P. stuartii in hospital-associated outbreaks which leads to excessive complications resulting in challenging treatments. Phage therapy is currently one of the most promising solutions to combat antibiotic-resistant infections. However, the number of available phages targeting Providencia spp. is extremely limited, restricting the use of phage therapy in such cases. In the present study, we describe the isolation and characterization of 17 lytic and temperate bacteriophages targeting clinical isolates of Providencia spp. as part of the Israeli Phage Bank (IPB). These phages, isolated from sewage samples, were evaluated for host range activity and effectively eradicated 95% of the tested bacterial strains isolated from different geographic locations and displaying a wide range of antibiotic resistance. Their lytic activity is demonstrated on agar plates, planktonic cultures, and biofilm formed in a catheter model. The results suggest that these bacteriophages can potentially be used for treatment of antibiotic-resistant Providencia spp. infections in general and of urinary tract infections in particular.

Phage therapy is a promising antibacterial strategy for resistant respiratory tract infections. Phage inhalation may serve this goal; however, it requires a careful assessment of their delivery by this approach. Here we present an in vitro model to evaluate phage inhalation. Eight phages, most of which target pathogens common in cystic fibrosis, were aerosolised by jet nebuliser and administered to a real-scale computed tomography-derived 3D airways model with a breathing simulator. Viable phage loads reaching the output of the nebuliser and the tracheal level of the model were determined and compared to the loaded amount. Phage inhalation resulted in a diverse range of titre reduction, primarily associated with the nebulisation process. No correlation was found between phage delivery to the phage physical or genomic dimensions. These findings highlight the need for tailored simulations of phage delivery, ideally by a patient-specific model in addition to proper phage matching, to increase the potential of phage therapy success.

Streptococcus mutans is a key bacterium in dental caries, one of the most prevalent chronic infectious diseases. Conventional treatment fails to specifically target the pathogenic bacteria, while tending to eradicate commensal bacteria. Thus, caries remains one of the most common and challenging diseases. Phage therapy, which involves the use of bacterial viruses as anti-bacterial agents, has been gaining interest worldwide. Nevertheless, to date, only a few phages have been isolated against S. mutans. In this study, we describe the isolation and characterization of a new S. mutans phage, termed SMHBZ8, from hundreds of human saliva samples that were collected, filtered, and screened. The SMHBZ8 genome was sequenced and analyzed, visualized by TEM, and its antibacterial properties were evaluated in various states. In addition, we tested the lytic efficacy of SMHBZ8 against S. mutans in a human cariogenic dentin model. The isolation and characterization of SMHBZ8 may be the first step towards developing a potential phage therapy for dental caries.