Chronic Suppression of a Multidrug-Resistant Pseudomonas aeruginosa in Prosthetic Joint Infection Using Personalized Bacteriophage Treatment

Chronic Suppression of a Multidrug-Resistant Pseudomonas aeruginosa in Prosthetic Joint Infection Using Personalized Bacteriophage Treatment | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Chronic Suppression of a Multidrug-Resistant Pseudomonas aeruginosa in Prosthetic Joint Infection Using Personalized Bacteriophage Treatment Kenneth Urish, Rekha Arya, Nadine Sadaka, Andrew Frear, Jewelia Rempuszewski, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5920650/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Multidrug resistant (MDR) bacterial infections without antibiotic options are a public health emergency. Infections associated with medical implants serve as an example. Conventional antibiotics have limited ability to eradicate these infections as they are associated with antibiotic-tolerant biofilms. Here, we report the use of bacteriophage therapy for the treatment of a MDR, non-operable Pseudomonas aeruginosa periprosthetic joint infection that had failed multiple antibiotic and surgical interventions. Treatment with intermittent bacteriophage therapy alone without antibiotics over a 2 year time period resulted in clinical resolution of the infection, but not microbiological eradication. Bacteriophage therapy established this control, in part, by altering bacterial virulence and disrupting biofilm. Whole genome sequencing demonstrated the continued presence of bacteriophage during treatment without integration into the bacteria genome. This provides preliminary evidence that bacteriophage can be used to treat multidrug-resistant infections when antibiotic options do not exist. Biological sciences/Microbiology/Bacteriophages Biological sciences/Molecular biology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Medical implants and devices have transformed medicine by providing a solution to a wide range of conditions that were previously impossible to treat. This has improved quality of life and preserved function while pushing the boundaries in a variety of surgical disciplines including cardiothorasic, vascular, dental, neurological, and orthopaedic fields. About half of all nosocomial infections are associated with medical devices 1 . These implant-associated infections are difficult to treat because of their association with antibiotic tolerant biofilm 2 . An example of this challenge includes total knee and hip arthroplasty (joint replacements). It is one of the most common surgeries in the developed world, and the most severe complication includes periprosthetic joint infections (PJI) 3 . It is a devastating diagnosis, often requiring multiple surgical procedures over several years with a five-year mortality between 20 to 25% 4,5 , higher than many cancers 5 . Periprosthetic joint infection is difficult to treat because of its association with antibiotic tolerant biofilms. Eradication of the infection requires the removal of components, but surgical intervention with implant removal can have significant morbidity or mortality. Periprosthetic joint infection is associated with multi-drug resistant (MDR) hospital-acquired ESKAPE organisms ( Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp , and Escherichia coli ). P. aeruginosa is a particularly challenging pathogen as conventional antibiotic treatments are often ineffective 6 , 7 . There is thus an urgent need to develop alternative therapies that can overcome the limitations of antimicrobial-based treatment strategies for implant-associated infections. Lytic bacteriophages, commonly known as phages, have the potential to be an effective therapy against these difficult-to-treat infections because of their innate anti-biofilm activity 8 , 9 . Phage therapy has been used as an adjuvant to antibiotics to treat PJI and other biofilm-associated infections 10 – 12 , 13 . A common strategy includes combining long-term conventional antibiotics with bacteriophage therapy to eradicate or suppress the complex infection. In this study, bacteriophage therapy was utilized in a unique way as a stand-alone therapeutic without antibiotics. A patient presented with a chronic multi-drug resistant P. aeruginosa PJI that was non-operative. They had been referred to palliative care after multiple surgical and antibiotic treatment failures. After standard of care treatment, the patient remained culture positive, but intermittent dosing of bacteriophage resulted in clinical control of the infection. This was achieved, in part, through long-term bacteriophage propagation, limiting biofilm formation, and a phenotypic decrease in bacteria virulence. Results Clinical Course and Bacteriophage Therapy A 96-year-old male with multiple comorbidities including heart failure presented with a chronic multi-drug resistant Pseudomonas aeruginosa infection in complex implants. A detailed clinical course and treatment protocol is provided (Fig. 1 a, Supplementary information ). Staged total knee and hip arthroplasties were performed more than 15 years previously that had been complicated by a fracture (Fig. 1 b-e). He was diagnosed with a P. aeruginosa PJI of his knee that metastasized to his ipsilateral hip (Fig. 1 f-g). Initially sensitive to oral antibiotics, the organism became multidrug resistant following chronic suppression with ciprofloxacin. Chronic suppression was no longer feasible given the bacteria was resistant to all oral antibiotic options. There were multiple attempts to discontinue intravenous antibiotics over an approximate 2 year period, but this resulted in painful symptomatic infection or sepsis. During each of these recurrences and multiple surgical interventions, the cultures continued to be positive for MDR Pseudomonas . The patient presented to our institution with a MDR Pseudomonas PJI and complex implants with limited treatment options. At the outside institution, intravenous antibiotics were planned to be stopped permanently with plans for palliative care and hospice. Traditional treatment options were limited including observation with discontinuation of antibiotics, repeat DAIR which had failed the previous four times, removal of components, or amputation. These options had a high probability of leaving him non-ambulatory and with a high expected mortality 5 , 14 . Given limited treatment options, FDA-expanded access was obtained to start bacteriophage therapy. Cultures were obtained and two bacteriophages (PASA16 and Φ83) 12 , 15 (Adaptive Phage Therapeutics (APT), Bethesda, MD) were identified with planktonic and biofilm activity against the P. aeruginosa clinical isolate tested. Titers of phage for dosing were selected based on maximizing phage titer below the threshold of endotoxin exposure (4.7 x 10 10 plaque-forming units (PFU)/mL PASA16 and 7.5 x 10 9 PFU/mL Φ83). Standard of care treatment was provided with debridement and antibiotics with implant retention (DAIR) using intermittent bacteriophage therapy as adjuvant care. The initial dose was intra-articular followed by a daily intravenous dose for one week. Diagnostic markers indicated continued infection despite clinical resolution The patient was transitioned to chronic suppression with intermittent bacteriophage treatment. Following standard of care treatment, knee aspiration was culture-negative, but continued to meet the Musculoskeletal Infection Society (MSIS) definition of infection 16 . Plaque assay on the synovial fluid demonstrated the continued presence and propagation of bacteriophage. The patient remained symptomatic with persistent knee effusion and pain at the site for 3 months. Intraarticular bacteriophage dosing was therefore repeated, and the effusion and pain subsided. Based on the initial success and catastrophic consequences of uncontrolled infection, bacteriophage was continued to be dosed at approximately every 4–6 months without antibiotics. Aspirations completed prior to dosing demonstrated that the patient remained culture positive (Fig. 1 a) and met the International Consensus Meeting (ICM) 2018 definition of prosthetic joint infection 17 (Fig. 2 a-c) but he continued to remain clinically asymptomatic. At 6 months, the knee effusion resolved. This corresponded with an overall decrease in inflammatory markers (Fig. 2 c, Supplementary Table 1 ). No significant adverse events were observed while on phage therapy. There was a transient elevation in liver enzymes which other groups have previously reported 12 (Fig. 2 d). The patient has remained off antibiotics and has remained asymptomatic at 2-year follow-up with baseline ambulatory function comparable to prior to infection. Long-term clinical control of the infection was established off of antibiotics for more than 2 years. Prior to use of bacteriophage therapy, discontinuation of antibiotics resulted in a painful leg with episodes of sepsis. Phage lytic activity against patient isolates over time Given the extended time period of treatment, phage activity in pre- and post-phage bacterial isolates was examined for phage sensitivity. For analysis, we selected pre-phage (S1 and S2), mid-treatment (S8), and long-term treatment (S22 and S23) pseudomonas isolates. The phages used for treatment, PASA16 ( Pbunavirus ) (Fig. 3 a, b) and Φ83 ( Podoviridae ) (Fig. 3 c, d), were assessed for activity using standard growth inhibition and a plaque assay (Supplementary Table 2) . For both PASA16 and Φ83, growth inhibition assays on pre-phage, mid-term, and long-term phage treatment isolates demonstrated no statistically significant loss of activity (Fig. 3 e, f). There was a morphological difference between the pre-phage and long-term treatment isolates obtained from the patient. The long-term treatment isolates exhibited a small colony variant phenotype ( Supplementary Fig. 1a, b ). Neutralizing antibody production Given the multiple doses, the patient’s host immunological response to bacteriophage therapy was investigated. Neutralizing antibodies have been observed in serum following intravenous phage therapy in multiple case reports 18 , 19 . As treatment progressed, we began to monitor for phage-neutralizing antibody activity initially in the serum and then in synovial fluid. Patient specific neutralizing antibodies to these phages were monitored by assessing changes in phage lytic activity when performed in serum or synovial fluid. For treatments involving PASA16, serum collected at 6, 9, 12 and 24 months showed a significant high neutralizing activity and reduced the PFU count ( P < 0.01; Fig. 3 g) when compared to the SM + buffer control. Incubation of the phage with patient synovial fluid showed a significant reduction ( P < 0.007, Fig. 3 h) in PFU count when compared to SM + control. We also compared the patient’s serum and synovial fluid against similar samples from an untreated patient. The results were similar, displaying diminished plaque formation in the patient’s samples as compared to those from unexposed serum and synovial fluid (Fig. 3 i, j). Comparable results were observed with Φ83 changes in activity from neautralizing antibodies observed in patient serum ( P < 0.001; Fig. 3 k) or synovial fluid ( P < 0.0005, Fig. 3 l), but not in unexposed serum and synovial fluid (Fig. 3 m, n). These findings reaffirm the presence of neutralizing antibodies within both serum and synovial fluid samples, displaying a consistent and marked reduction in viral activity over time when challenged with Φ83 and PASA16. This response pattern suggests bacteriophage therapy maintained an active suppression of the infection in the presence of an immunological response from the patient’s neutralizing serum and synovial antibodies. Bacteriophages reduce and eradicate patient P. aeruginosa biofilm Control of the biofilm associated implant infection suggested the phages used had strong anti-biofilm activity. We hypothesized that bacteriophage therapy could reduce the formation of and potentially eradicate P. aeruginosa biofilm. We saw a notable impact of bacteriophages Φ83 and PASA16 on biofilm formation in P. aeruginosa clinical isolates collected at different time points. Utilizing both inhibition and eradication assays, concurrent incubation with these bacteriophages led to a significant reduction in biofilm formation compared to non-treated controls. In the inhibition assay, a substantial decrease in biofilm formation was observed. Pre-phage samples S1 and S2 treated with PASA16 and Φ83 resulted in a significant decrease in biofilm formation ( P = 0.0001) compared to untreated controls. Mid-term samples (S8, S13) and late treated isolates (S22, S23) had statistically reduced biofilm formation when treated with PASA16 and Φ83 as compared to untreated controls ( P = 0.0001; Fig. 4 a ) . Biofilms have a high tolerance to antibiotics 20 . Based on these results, we hypothesized that bacteriophages would have strong anti-biofilm activity. Biofilm was cultured using the clinical isolates, treated with bacteriophage, and remaining biofilm was quantified. Pre (S1, S2), mid-term (S8,13), and late treated (S22,S23) isolates had a significant decrease in biofilm following treatment as compared to untreated controls ( P < 0.01; Fig. 4 b ) . These results demonstrate a robust capability of both bacteriophages in inhibiting and eradicating biofilms of P. aeruginosa isolates, and were supported by qualitative observations with Crystal Violet staining. These experiments were repeated with antibiotics. Biofilm were treated with 5x, 20x and 50x MIC of ciprofloxacin (CIP), cefepime (FEP), meropenem (MEM), and piperacillin/tazobactam (P/T). Biofilm remained following treatment ( Supplementary Fig. 2). Phage therapy reduces P. aeruginosa virulence We hypothesized that bacteriophage treatment may have altered bacteria virulence. The nematode Caenorhabditis elegans infection model was used as a measure of global virulence (Fig. 5 a). Serial passage was generated with phage growth inhibition assays and was compared with patient isolates for virulence inhibition. Pseudomonas overgrowth from the initial assay was used as the inoculation for a repeat growth inhibition assay. At each repeat passage, phage growth inhibition remained unchanged demonstrating that the bacteria isolate had not developed a phage resistance genotype. C. elegans survival assays were performed using the patient isolates and pseudomonas isolate S1 from the serial growth inhibition assays using corresponding PASA16 and Φ83 (termed S1PASA16 OG and S1Φ83 OG ; Fig. 5 b, c). C. elegans infected with serial phage overgrowth pseudomonas (S1PASA16 OG and S1Φ83 OG ) had a comparable survival as pseudomonas S1 that had not been exposed to bacteriophage ( Supplementary Fig. 3a-f) . To test other metrics of virulence, swarming ( Supplementary Fig. 4a-f ) and swimming motility ( Supplementary Fig. 5a-f ) were measured. Comparing pseudomonas unexposed to phage (S1) to pseudomonas overgrowth from phage (S1PASA16 R and S1Φ83 R ), swarming (Fig. 5 d, e), swimming (Fig. 5 f, g), and twitching ( Supplementary Fig. 6a-f ) was similar as unexposed isolates. Genomic analysis of patient P. aeruginosa isolates Given the patient’s continued positive cultures but overall control of the infection, genomic variation was assessed to quantify any genotype changes in the pseudomonas isolates. Selective pressures from different local environments of the infection can drive adaptive mutations 21 . Based on pre-and post-treatment with phages and antibiotic susceptibilities patterns ( Supplementary Table 3 ), twenty-seven isolates (pre-phage: S1, S2, S3, S4, S5, S6 and S7; long-term treatment: S8, S9, S10, S11, S12, S13, S14, S15, S16, S17, S18, S19, S20, S21, S22, S23, S24, S25, S26 and S27) were selected for whole genome sequencing (WGS). Each longitudinal isolate was compared with the assembled and annotated genome of the first patient isolate to identify mutations arising during the infection and/or associated with phage therapy (Table 1 ). Nineteen of these isolates were genetically identical to the initial isolate, whereas eight isolates acquired 1–3 new mutations of unknown relevance to the infection or phage treatment. These findings demonstrate limited evolution of the P. aeruginosa population during this infection that was controlled by bacteriophage therapy (Table 2 ). These results also showed that mutations and pseudogenes occurred in pre-mid and post-phage treated samples, but these mutations were not caused by the post-phage treatment and are not consistently present in post-phage treated samples (as shown in Supplementary Fig. 7a, b ). WGS data revealed that phages were present on circularized contigs, and the read coverage profile is inconsistent with that of the bacterial genome. Therefore, it indicates that phage genes were present on aspiration but not integrated into the bacterial genome. Discussion Antibiotic resistance is a public health emergency. Multi-drug resistant medical device-related infections illustrate these complex challenges. Here we demonstrate that bacteriophage therapy alone could be used to chronically suppress a multi-drug resistant P. aeruginosa infection that had failed multiple surgical and antibiotic courses. Clinical control of the infection was established, in part, by bacteriophage modulating bacteria virulence and its activity against biofilm. Biofilms are associated with medical device infections and are highly tolerant to antibiotics 20,22,23 3 as demonstrated by continued treatment failure despite prolonged extended intravenous antibiotics. Bacteriophage has been demonstrated in vitro to have biofilm activity in other organisms 24,25 , but there is no previous demonstration of possible clinical efficacy. To address this, we tested the efficiency of our bacteriophages on biofilms of the clinical isolates. The results suggest that the bacteriophages reduced and limited P. aeruginosa biofilm during clinical treatment. After bacteriophage therapy, a phenotypic decrease in P. aeruginosa virulence was observed. This was observed by a statistically significant increase in C. elegans survival and reduced motility after long-term phage treatment. A loss of virulence resulted in an ability for the bacteriophage to develop clinical control of the infection without eradication. These changes were not from host adaptation. The pre-phage isolates were an already host-adapted clone that colonized the patient for several years. As well, there was minimal genotype variation between clinical Pseudomonas isolates before and after phage treatment. No phage integration was observed. Host antibodies were observed directed to the bacteriophage, but continued clinical utility continued to be observed. This has been reported in other case reports 26,27 . These findings suggest that the use of bacteriophage therapy resulted in the clinical control of the infection without eradication secondary to phage biofilm activity and a phenotypic alteration in bacteria virulence. This demonstrates an important clinical concept for the ability of bacteriophage to be used in chronic suppression of implant-associated infections. When an antibiotic option does not exist with multidrug-resistant bacteria, our data provides preliminary evidence that bacteriophage may be an alternative therapy. This supports conducting prospective clinical studies. Methods Ethical statements The patient has consented to our use of samples and data in research and to the publication of clinical data, the images presented here. He also gave written informed consent to receive phage therapy in accordance with CARE guidelines. For phage therapy, patient cultures were sent to phage company (Adaptive Phage Therapy) and a phage match was obtained. Once phage match was found, we obtained compassionate-use IRB (EA21110067) from the University of Pittsburgh and FDA approval under expanded access (IND 28197). Bacterial strains, bacteriophage and growth conditions Synovial fluid and blood samples were collected from the patient during clinical visits. Serum was extracted from blood samples and stored as frozen aliquots at – 80 °C. P. aeruginosa isolates, collected from the patient’s cultures, were provided by the UPMC Presbyterian Microbiology Lab. Bacteriophages, Φ83 and PASA16, their host cell, C10, and the original patient cultures, S1 and S2, were provided by Adaptive Phage Therapeutics (APT). All bacterial strains were grown in Mueller Hinton Broth (MHB; Bectin Dickinson and Company) medium with shaking at 37 °C and stored as frozen stocks in 30% glycerol at – 80 °C. All phages were tested using a standard plaque assay, expanded, purified and stored as frozen stocks in 30% glycerol at – 80 °C. Growth inhibition assay S1, S2, S8, S13, S22, and S23 were inoculated into 5 ml MHB medium and grown overnight at 37ºC. Cultures were normalized to 5x10 5 CFU/mL using a 0.5× McFarland turbidity standard (Hardy Diagnostics). The bacteriophages, Φ83 and PASA16, were tittered to approximately 1x10 9 CFU/ml using a standard plaque assay. Cultures were plated in triplicates in 96 well plates (Costar) and bacteriophages were added and incubated at 37 °C for 24 h at shanking incubator. After 24 h the optical density was observed in the microplate reader at 600nm (BioTek). Plaque Assay To perform the experiment, 100 μL of each clinical isolate's overnight culture was mixed with 10mL of pre-warmed lysogeny broth (LB) containing 0.75% (w/v) of agar (molten agar) and 5mM of CaCl 2 . This mixture was then poured into 90mm Petri dishes and allowed to solidify. Once the agar had solidified, 10μL drops of PASA16 and Φ83 phage stock, which had been serially diluted ten times, were placed on top of the agar. The drops were left to dry at room temperature and the Petri dishes were then incubated at 37°C overnight. The next day, the plates were checked for lysis zones and the PFU were calculated. Minimum inhibitory concentration (MIC) Cefepime, Ciprofloxacin, Meropenem, and Zosyn minimum inhibitory concentrations for all patient cultures were determined by UPMC Presbyterian Microbiology Lab. Phage-targeting antibody neutralization assay Using a standard plaque assay, high-titer lysates of Φ 83 and PASA 16 were created. Patient serum samples from pre-phage and post-phage time points were thawed on ice. Serum samples collected from 3 different timepoints were added to each Φ83 and PASA16 in sterile Eppendorf tubes. SM+ buffer was used as a control. Serum and phage were mixed thoroughly, and then incubated at room temperature overnight. After 24 h, serial dilutions of each mixture were performed in SM+ buffer. Then, 5 uL of each dilution was plated in triplicates onto a top agar lawn of C10. The agar plate was incubated at 37 °C for 24 h. Plaques were counted, and the presence of neutralizing activity was determined in PFU/mL. Additionally, to confirm the presence of neutralizing antibodies this assay was repeated to compare the patient’s serum and synovial fluid samples to control human serum and synovial fluid samples. Titanium rod biofilm MIC assay Titanium rods (6 mm) were placed into a 48-well tissue culture plate containing 20 wells of MHB with 1x10 6 CFU/ml P1. The plate was incubated at 37 °C for 48 h. To remove non-adherent cells, wells were replaced with fresh MHB after 24 h. After 48 h, titanium rods were washed in 1 ml PBS and placed into a 48-well tissue culture plate containing Cefepime, Ciprofloxacin, Meropenem, and Zosyn at 1x, 5x, 20x, and 50x MIC. The plate was incubated at 37 °C for 24 h. After 24 h, titanium rods were washed in 1 ml of PBS containing 1% tween 20 (PBST). To break up the biofilm, samples were sonicated using a Branson model #3510 sonicating water bath for 10 min. Serial dilutions of the sonicated bacterial suspension were prepared and plated onto blood agar plates and incubated overnight at 37 °C. Colonies were counted, and the biofilm bacterial burden was determined in CFU/mL. Assay was repeated for S2, S8, S13, S22, and S23. Biofilm inhibition assay S1, S2, S8, S13, S22, and S23 were inoculated into 5 ml MHB medium and grown overnight at 37ºC. Cultures were normalized to 1x10 6 CFU/ml using a 0.5× McFarland turbidity standard (Hardy Diagnostics) and bacteriophages, Φ83 and PASA16, were tittered to approximately 1x10 8 CFU/ml using a standard plaque assay. Phage were added to the cultures to achieve multiplicity of infection (MOI) of 1:100 and plated in triplicates in 96 well plates (Costar) with untreated bacteria as the control. The plate was incubated at 37 °C for 48 h. After incubation, non-adherent cells were removed by turning the plate over and shaking out the liquid. The plate was gently submerged in a small tub of water and then shaken out. Using a micropipette, 200 uL of water were gently added to the wells and then shaken out; this process was repeated 3-4x. Next, 125 uL of crystal violet (CV) was added to the wells and incubated at room temperature for 10-15 minutes. The wells were gently rinsed 3-4x and left to dry upside down in laminar flow hood for 10-15 minutes. To solubilize the CV, 125 uL of 30% acetic acid was added to the wells and incubated at room temperature for 10-15 minutes. To assess biofilm inhibition, absorbance was quantified in a plate reader at 550 nm using 30% acetic acid as the blank. For qualitative purposes, plates were photographed. Biofilm eradication assay S1, S2, S8, S13, S22, and S23 were inoculated into 5 mL MHB medium and grown overnight at 37ºC. Cultures were normalized to 1x106 CFU/ml using a 0.5× McFarland turbidity standard (Hardy Diagnostics) and bacteriophages, Φ83 and PASA16, were tittered to approximately 1x10 8 CFU/ml using a standard plaque assay. Cultures were plated in triplicates in 96 well plates (Costar) and incubated at 37 °C for 48 h. After 48 h, Φ83 and PASA16 were added to the wells and leaving 3 wells untreated as the control. The plate was incubated at 37 °C for 28 h. After incubation, non-adherent cells were removed by turning the plate over and shaking out the liquid. The plate was gently submerged in a small tub of water and then shaken out. Using a micropipette, 200 uL of water were gently added to the wells and then shaken out; this process was repeated 3-4x. Next, 125 uL of crystal violet (CV) was added to the wells and incubated at room temperature for 10-15 minutes. The wells were gently rinsed 3-4x and left to dry upside down in laminar flow hood for 10-15 minutes. To solubilize the CV, 125 uL of 30% acetic acid was added to the wells and incubated at room temperature for 10-15 minutes. To assess biofilm eradication, absorbance was quantified in a plate reader at 550 nm using 30% acetic acid as the blank. For qualitative purposes, plates were photographed. Serial passage A single colony of the clinical isolate S1 was grown overnight in LB medium on an orbital shaker at 180 rpm for 37°C. Afterward, 100 µL of the overnight culture was diluted in fresh LB medium to maintain a culture of 5 x 10 5 CFU/mL. This culture was then seeded in a 24-well plate, where PASA16 and Φ83 were added to each well, and the phage count was maintained at 5 x 10 7 PFU/mL. The next day, each population was serially propagated by transferring 25 µL of the overnight culture to 1.5 mL of fresh LB medium containing phages. This process was repeated for 10 days. Following the serial passages, we plated the culture on LB plates and counted the CFU the next day. When PASA16 was added to the S1 clinical isolate, the resulting adaptive evolution strain was designated S1PASA16 R . Similarly, when Φ83 was added to the S1 clinical isolate, the adaptive evolution strain was named S1Φ83 R . A single colony from these cultures was then picked, allowed to grow in LB medium, and frozen at -80°C in glycerol for future use. This culture was further utilized in experiments to assess C. elegans survival, as well as swarming and swimming motility. C. elegans maintenance The wild type C. elegans (N2 strain) was purchased from Carolina Biological Supply in Burlington, NC, USA for use in this study. The nematodes were grown and propagated on nematode growth media, which consisted of 15g agar, 2.4g NaCl 2 , 2g tryptone, 2.72 g KH 2 PO 4 , 0.8 mL of 1 M CaCl 2 , 5mg/mL of cholesterol, and 1 M MgSO 4 per L. The nematodes were fed with E. coli OP50 and kept at 25 O C 28 . To synchronize the worms, 5M NaOH and 5% bleach were used, and eggs were collected. The eggs were washed with M9 medium, which contains 6g Na 2 HPO 4 , 3g KH 2 PO 4 , 5g NaCl, and 0.25 g MgSO 4 ∙7H 2 O. The eggs were then incubated with S-complete medium (S-basal medium: 5.9 g NaCl, 50 mL of 1M potassium phosphate, pH 6.0 in 1L; S-complete medium: 977 mL S-basal, 10 mL 1M potassium citrate pH 6.0, 10 mL Trace metals solution, 3 ml 1M CaCl 2 , 3 mL 1M MgSO 4 ) to allow hatching. For the experiment, age-synchronized L4 worms were incubated in 96-well plates containing 100μL of S-complete medium in each well. C. elegans in vivo infection model For the infection assay, 96-well plate was filled with S-complete medium added with overnight culture of E. coli OP50. Then 15 mature L4 stage C. elegans were transferred in to medium. The total volume in the 96 well plate was maintained at 100 μL. Next, clinical isolates ( P. aeruginosa ) were grown overnight and washed with PBS and maintain the 1x10 6 CFU/mL for infection. The efficacy of phage therapy was studies using concentration of bacteria and phage with 1:100 multiplicity of infection (MOI). The Group 1 (control) consisted of S-complete medium with 15 nematodes, no bacteria. Group 2 (vehicle control): S-complete medium with 15 nematodes, no bacteria, PBS. Group 3 (infection control): S-complete medium with 15 nematodes infected with S1. Group 4 (phage treatment): S-complete medium with 15 nematodes infected with S1 and treated with PASA16. Group 5 (phage treatment): S-complete medium with 15 nematodes infected with S1 and treated with Φ83. Group 6 (infection control): S-complete medium with 15 nematodes infected with S2. Group 7 (phage treatment): S-complete medium with 15 nematodes infected with S2 and treated with PASA16. Group 8 (phage treatment): S-complete medium with 15 nematodes infected with S2 and treated with Φ83. Group 9 (infection control): S-complete medium with 15 nematodes infected with S3. Group 10 (phage treatment): S-complete medium with 15 nematodes infected with S3 and treated with PASA16. Group 11 (phage treatment): S-complete medium with 15 nematodes infected with S3 and treated with Φ83. Group 12 (infection control): S-complete medium with 15 nematodes infected with S4. Group 13 (phage treatment): S-complete medium with 15 nematodes infected with S4 and treated with PASA16. Group 14 (phage treatment): S-complete medium with 15 nematodes infected with S4 and treated with Φ83. Group 15 (infection control): S-complete medium with 15 nematodes infected with S5. Group 16 (phage treatment): S-complete medium with 15 nematodes infected with S5 and treated with PASA16. Group 17 (phage treatment): S-complete medium with 15 nematodes infected with S5 and treated with Φ83. Group 18 (infection control): S-complete medium with 15 nematodes infected with S6. Group 19 (phage treatment): S-complete medium with 15 nematodes infected with S6 and treated with PASA16. Group 20 (phage treatment): S-complete medium with 15 nematodes infected with S and treated with Φ83. The plates were incubated at 20 O C, and survival was monitored every 24 h for 7 days. The results were observed and evaluated based on live and dead C. elegans . All the experiments were repeated a minimum of three times for statistical significance. Motility assays Swarming Motility Assay Swarming motility was tested as reported previously with a few modifications 29 . To create the swimming plates, 6.5 g of agar was added to 800 mL of water, and the mixture was then autoclaved. 200 mL of a 5× M8 solution was then added to the agar mixture. The 5× M8 solution was created in advance by combining 30 g Na 2 HPO 4 , 15 g KH 2 PO 4 , and 2.5 g of NaCl with enough water to obtain a final volume of 1 L. After the addition of the 5× M8 solution, 10 mL of 20% glucose, 25 mL of 20% casamino acids, and 1 mL of 1 M MgSO 4 were combined with the agar solution. The solution was mixed and cooled prior to being poured in petri dishes (〜25 mL/plate), and the plates were allowed to solidify overnight at room temperature. For the experimental test plates, 10 μL of an overnight bacterial culture from a clinical isolate was combined with 10 μL of the phage being tested (PASA16 or ɸ83). This step was repeated for each of the 6 clinical samples being tested. The plates were then inoculated with 2.5 μL of bacteria, or 2.5 μL of the bacteria and phage mixture, and incubated upright for 24 hours. Following incubation, the zone diameter was recorded for each plate. Swimming Motility Assay Swimming motility was analyzed as previously described with minor modifications 29 . Plates were created as previously described for the Swarming Motility Assay, but 3 g of agar was used. For the experimental test plates, 10 μL of an overnight bacterial culture from a clinical isolate was combined with 10 μL of the phage being tested (PASA16 or ɸ83). A toothpick was dipped into this mixture, and stabbed partway into the agar plates. The plates were then incubated upright for 24 hours, and the zone diameter was measured. Twitching Motility Assay Twitching motility was accessed as previously described with minor modifications 30 . LB agar plates were stab inoculated with a mixture of 10 μL of an overnight bacterial culture from clinical samples and 10 μL of the phage being tested (PASA16 or ɸ83). The plates were then incubated for 48 hours and the agar was removed. The remaining attached cells were stained with a 1% Crystal violet solution, and the size of twitching was then measured. Statistical analysis All graphical and statistical analysis was performed using Prism 9.5 (GraphPad, La Jolla, CA). Statistical analysis was performed using one-way ANOVA with multiple comparisons test for the neutralizing antibodies data, and an unpaired t-test for the biofilm data. Significance was determined at p < 0.05. Images were treated with ImageJ (v1.53). Whole-genome sequencing and Raw read processing DNA from twenty-seven P. aeruginosa isolates was extracted using Qiagen kit according to the manufacturer’s protocol. Briefly, genomic DNA was isolated from frozen cell pellets using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) specifically tailored for the QIAcube. The elution process was conducted over a 10-minute period into nuclease-free water to ensure the purity of the DNA. The concentration of the extracted DNA was determined using the Qubit® 3.0 Fluorometer. Later, sample was sent to SeqCoast Genomics for further analysis. Trimmomatic (version 0.39) was used with the following parameters to quality-trim reads based on phred33 quality scores and remove adaptor sequences 31 . Genomes were assembled using the Unicycler software (version 0.4.4) 32 . Annotation was performed using BAKTA (version 1.6.1) 33 . Comparative genomics was performed using DIAMOND (version 2.0.15.153, Buchfink et al ., 2015) and the Pseudofinder software 34 . Variant-calling was performed using the Breseq software (version 0.36) 35 . Breseq was run with the consensus-minimum-variant-coverage-each-strand flag set to 3, and all other default parameters. Sample 1 was used as the reference genome, and false-positive mutations were filtered out based on their presence in sample 1. Principal component analysis was performed using the R package factoextra. Declarations Acknowledgements This work would not have been possible without the support of Robert Hopkins, MD, Kendra Weaver, Acknowledgements Reddy, Anantha Makineni, Aravinda Vadlamudi, Jordan Erickson, Laura Hauns. The funders had no role in shaping the conceptual framework, designing the study, collecting or analyzing data, deciding to publish, or preparing the manuscript. Author contributions All authors contributed to the data analysis and outlined the study; R.A. and K.L.U designed the experiments. R.A., N.M.S., A.J.F. and J.J.R. conducted most of the studies; V.S.C. conducted the bioinformatic analysis and analysed the data; R.A., K.L.U wrote the manuscript with contributions from other authors. Competing interests The authors declare no competing interests. Correspondence and requests for materials should be addressed to Kenneth L. Urish References Darouiche, R.O.J.N.E.J.o.M. Treatment of infections associated with surgical implants. 350 , 1422-1429 (2004). Hall-Stoodley, L., Costerton, J.W. & Stoodley, P.J.N.r.m. Bacterial biofilms: from the natural environment to infectious diseases. 2 , 95-108 (2004). Bozic, K.J. , et al. The epidemiology of revision total knee arthroplasty in the United States. 468 , 45-51 (2010). Shah, N.B. , et al. Benefits and adverse events associated with extended antibiotic use in total knee arthroplasty periprosthetic joint infection. 70 , 559-565 (2020). Drain, N.P. , et al. High mortality after total knee arthroplasty periprosthetic joint infection is related to preoperative morbidity and the disease process but not treatment. 37 , 1383-1389 (2022). Perez-Jorge, C., Gomez-Barrena, E., Horcajada, J.-P., Puig-Verdie, L. & Esteban, J.J.E.O.o.P. Drug treatments for prosthetic joint infections in the era of multidrug resistance. 17 , 1233-1246 (2016). Sultan, M., Arya, R. & Kim, K.K.J.I.j.o.m.s. Roles of two-component systems in Pseudomonas aeruginosa virulence. 22 , 12152 (2021). Brives, C. & Pourraz, J.J.P.C. Phage therapy as a potential solution in the fight against AMR: obstacles and possible futures. 6 , 1-11 (2020). York, A.J.N.R.M. Phages to the rescue. 20 , 703-703 (2022). Pirnay, J.-P. , et al. Personalized bacteriophage therapy outcomes for 100 consecutive cases: a multicentre, multinational, retrospective observational study. 1-20 (2024). Doub, J.B. , et al. Experience using adjuvant bacteriophage therapy for the treatment of 10 recalcitrant periprosthetic joint infections: a case series. 76 , e1463-e1466 (2023). Onallah, H. , et al. Refractory Pseudomonas aeruginosa infections treated with phage PASA16: A compassionate use case series. 4 , 600-611. e604 (2023). Tkhilaishvili, T. , et al. Bacteriophages as adjuvant to antibiotics for the treatment of periprosthetic joint infection caused by multidrug-resistant Pseudomonas aeruginosa. 64 , 10.1128/aac. 00924-00919 (2019). Migliorini, F. , et al. Bacterial pathogens and in-hospital mortality in revision surgery for periprosthetic joint infection of the hip and knee: analysis of 346 patients. 28 , 177 (2023). Alkalay-Oren, S. , et al. Complete genome sequence of Pseudomonas aeruginosa bacteriophage PASA16, used in multiple phage therapy treatments globally. 11 , e00092-00022 (2022). Dombrowski, M.E., Klatt, B.A., Deirmengian, C.A., Brause, B.D. & Chen, A.F.J.I.C.L. Musculoskeletal Infection Society (MSIS) Update on Infection in Arthroplasty. 69 , 85-102 (2020). Parvizi, J. , et al. The 2018 definition of periprosthetic hip and knee infection: an evidence-based and validated criteria. 33 , 1309-1314. e1302 (2018). Berkson, J.D. , et al. Phage-specific immunity impairs efficacy of bacteriophage targeting Vancomycin Resistant Enterococcus in a murine model. 15 , 2993 (2024). Dedrick, R.M. , et al. Phage therapy of Mycobacterium infections: compassionate use of phages in 20 patients with drug-resistant mycobacterial disease. 76 , 103-112 (2023). Ma, D. , et al. The toxin-antitoxin MazEF drives Staphylococcus aureus biofilm formation, antibiotic tolerance, and chronic infection. 10 , 10.1128/mbio. 01658-01619 (2019). Ma, D. , et al. Staphylococcus aureus genotype variation among and within periprosthetic joint infections. 40 , 420-428 (2022). Ma, D. , et al. Viable bacteria persist on antibiotic spacers following two‐stage revision for periprosthetic joint infection. 36 , 452-458 (2018). Urish, K.L. , et al. Antibiotic-tolerant Staphylococcus aureus biofilm persists on arthroplasty materials. 474 , 1649-1656 (2016). El-Atrees, D.M., El-Kased, R.F., Abbas, A.M. & Yassien, M.A.J.S.R. Characterization and anti-biofilm activity of bacteriophages against urinary tract Enterococcus faecalis isolates. 12 , 13048 (2022). Abdelghafar, A., El-Ganiny, A., Shaker, G., Askoura, M.J.E.J.o.C.M. & Diseases, I. A novel lytic phage exhibiting a remarkable in vivo therapeutic potential and higher antibiofilm activity against Pseudomonas aeruginosa. 42 , 1207-1234 (2023). Viazis, S., Akhtar, M., Feirtag, J., Brabban, A. & Diez‐Gonzalez, F.J.J.o.A.M. Isolation and characterization of lytic bacteriophages against enterohaemorrhagic Escherichia coli. 110 , 1323-1331 (2011). Sattar, S. , et al. Characterization of two novel lytic bacteriophages having lysis potential against MDR avian pathogenic Escherichia coli strains of zoonotic potential. 13 , 10043 (2023). Chaudhuri, J., Parihar, M. & Pires-daSilva, A.J.J. An introduction to worm lab: from culturing worms to mutagenesis. e2293 (2011). Filloux, A. & Ramos, J.-L. Methods and Protocols. Déziel, E., Comeau, Y. & Villemur, R.J.J.o.b. Initiation of biofilm formation by Pseudomonas aeruginosa 57RP correlates with emergence of hyperpiliated and highly adherent phenotypic variants deficient in swimming, swarming, and twitching motilities. 183 , 1195-1204 (2001). Bolger, A.M., Lohse, M. & Usadel, B.J.B. Trimmomatic: a flexible trimmer for Illumina sequence data. 30 , 2114-2120 (2014). Wick, R.R., Judd, L.M., Gorrie, C.L. & Holt, K.E.J.P.c.b. Unicycler: resolving bacterial genome assemblies from short and long sequencing reads. 13 , e1005595 (2017). Schwengers, O. , et al. Bakta: rapid and standardized annotation of bacterial genomes via alignment-free sequence identification. 7 , 000685 (2021). Syberg-Olsen, M.J. , et al. Pseudofinder: detection of pseudogenes in prokaryotic genomes. 39 , msac153 (2022). Barrick, J.E. , et al. Identifying structural variation in haploid microbial genomes from short-read resequencing data using breseq. 15 , 1-17 (2014). Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryfileWBNATURECOMMUNICATIONS.docx Supplementary file Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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\u003c/em\u003e\u003cem\u003e\u003cstrong\u003ea,\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Clinical timeline of PJI course by months. M0 designates time of first bacteriophage therapy. Bacteriophage therapy (black), oral antibiotic treatment (purple), intravenous \u003c/em\u003epiperacillin-tazobactam \u003cem\u003e(blue), intravenous cefepime (yellow), fluoroquinolone \u003c/em\u003e(\u003cem\u003eFQ). Radiographs demonstrate complexity of implants and hardware. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eb, \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eAnterior-posterior supine pelvis radiograph; \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ec\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e, Left lateral femur radiograph; \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ed\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e, Anterior-posterior standing knee radiograph; \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ee\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e, Lateral left knee extension radiograph; PET imaging demonstrated PJI of entire femur: \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ef\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e, Anterior-Posterior PET imaging of the pelvis and upper leg; \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eg\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e, Lateral PET-CT of left upper.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5920650/v1/45cdefb043190a0ddeedb295.png"},{"id":75709175,"identity":"ac53b792-10e5-4bb3-b139-56ded852970c","added_by":"auto","created_at":"2025-02-07 10:49:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":191698,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eClinical laboratory values measured at different time points (0, 3, 4, 9, 12, 14, 17, 21 and 26 months) following the initiation of phage therapy. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e, knee aspirate with nucleated cell counts and differential; half-shaded orange circles and red squares indicate moderate or minimal effusion beginning at 14-months post initial phage therapy. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e, hip aspirate data; the asterisks, open orange circles and red squares show the absence of effusion. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ec\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e, inflammatory serology; erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) levels, with open blue circles and purple squares representing no reported pain by the patient, starting after the 12-months. Effusion resolved at 6-9 months post-initial treatment as represented by the shading. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ed\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e, liver function displaying AST and ALT levels, with thresholds marked at ≤ 40 U/L for normal values. Dotted horizontal lines in all graphs represent the elevated threshold for Musculoskeletal Infection Society (MSIS) and International Consensus Meeting (ICM 2018) criteria for prosthetic joint infection (PJI) including nucleated cell count \u0026lt;3000 cells/mm³, neutrophils \u0026lt;80%, ESR 0-23 mm/h, and CRP \u0026lt;0.8 mg/dL.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5920650/v1/a96baa23554a5f5c9c69c62a.png"},{"id":75709176,"identity":"beecc141-d3de-41c4-bbfb-571c6279a4b4","added_by":"auto","created_at":"2025-02-07 10:49:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":343520,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePhage lytic activity on pre-, mid- and late-term treatment samples\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e. a\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e, PASA16 visualized by TEM\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e. b\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e, PASA16 bacteriolytic activity on host strain C10; and on patient pre, mid and late-term treated isolates\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e. c\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e, Φ83 Visualized by TEM\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e. d\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e, Φ83 bacteriolytic activity in host strain C10; and in patient pre-, mid and late-term treated isolates. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ee\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e, Inhibition of planktonic growth by PASA16 on Pre- mid and late-term treated isolates. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ef\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e, Inhibition of planktonic growth by Φ83 on Pre- mid and late-term treated isolates. The presence of neutralizing antibodies against bacteriophages PASA 16 and Φ83 in patient serum and synovial fluid samples. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eg,\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e PASA16 activity in SM+ buffer and serum samples from different time points (6, 9, 12 and 24 months). \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eh\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e, PASA16 activity in synovial fluid sample. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ei, j\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e, PASA16 mixed with SM+ buffer, non-patient human serum, synovial fluid and compared with phage patient sample. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ek\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e, Φ83 activity in SM+ buffer and serum samples from different timepoints (6, 9, 12 and 24 months). \u003c/em\u003e\u003cem\u003e\u003cstrong\u003el\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e, Φ83 activity in synovial fluid sample. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003em,n,\u003c/strong\u003e\u003c/em\u003e\u003cem\u003eΦ83 mixed with SM+ buffer, non-patient human serum sample, synovial fluid sample and compared with phage patient samples. Values are presented as mean ± SEM of three independent experiments performed in technical replicates.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5920650/v1/45428e4d8338b942316260fc.png"},{"id":75708750,"identity":"5be98dd4-12d4-4ea5-bcfd-5485a2760e37","added_by":"auto","created_at":"2025-02-07 10:41:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":267297,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eBiofilm formation and eradication in patient-derived Pseudomonas aeruginosa isolates. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e, PASA16 and Φ83 inhibiting P. aeruginosa biofilms (bacteria and phage ratio of 1:100, i.e., 10\u003c/em\u003e\u003csup\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e CFU/mL and 10\u003c/em\u003e\u003csup\u003e\u003cem\u003e8\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e PFU/mL), in the pre-phage sample (S1, S2),\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cem\u003emid-term (S8, S13) and late-term treatment samples (S22 and S23). \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e, PASA16 and Φ83 phages in eradicating P. aeruginosa biofilms (bacteria and phage ratio of 1:100, i.e., 10\u003c/em\u003e\u003csup\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e CFU/mL and 10\u003c/em\u003e\u003csup\u003e\u003cem\u003e8\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e PFU/mL) in the pre-phage sample (S1, S2), mid-term (S8, S13) and late-term treatment samples (S22 and S23). Photographic evidence of the Crystal Violet-stained biofilms provides qualitative validation. Experiments performed in replicate.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5920650/v1/b072e8d97ad9f8ac3c9b4b8c.png"},{"id":75710781,"identity":"2d01e437-b8bd-4c73-92cf-63b3095c3170","added_by":"auto","created_at":"2025-02-07 11:05:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":234985,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePhage therapy in C. elegans infected with patient isolates and compared with serial passage strain. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e, Schematic representation of experimental design; \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eb, \u003c/strong\u003e\u003c/em\u003e\u003cem\u003e\u0026nbsp;Representative survival of C. elegans infected by pre-, mid- and long-term samples and treated with PASA16 and Φ83; \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ec, \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eSurvival of C. elegans infected with serial passage strain S1 generated by PASA16 and Φ83 growth inhibition assay \u003c/em\u003e(\u003cem\u003eS1PASA16\u003c/em\u003e\u003csup\u003e\u003cem\u003eOG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e and S1Φ83\u003c/em\u003e\u003csup\u003e\u003cem\u003eOG\u003c/em\u003e\u003c/sup\u003e) \u003cem\u003eand treated with PASA16 and Φ83.\u003c/em\u003e \u003cem\u003eBacteria and phage ratio of 1:100, i.e., 10\u003c/em\u003e\u003csup\u003e\u003cem\u003e5\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e CFU/mL and 10\u003c/em\u003e\u003csup\u003e\u003cem\u003e7\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e PFU/mL. Effect of bacteriophage on swarming in P. aeruginosa clinical isolates. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e\u0026nbsp;d\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e, Pre-, mid- and long-term\u0026nbsp; isolates were treated with PASA16, Φ83 and swarming were analysed after 16 h at 37°C and zone diameter was measured in millimetres. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ee, \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eSwarming was assessed in sample S1 generated through serial passage and treated with PASA16 and Φ83. Effect of bacteriophage on swimming in P. aeruginosa clinical isolates\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e. f,\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Pre- mid- and long-term isolates were \u0026nbsp;treated with PASA16, Φ83 and swimming were analysed after 16 h at 37°C and zone diameter was measured in millimetres. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eg\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e, Swimming was analyzed in sample S1 generated through serial passage and treated with PASA16 and Φ83. All the experiments were performed in replicates and asterisks indicate significant differences between the means and the control at p \u0026lt; 0.05 by analysis of variance followed by unpaired t-test.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5920650/v1/70e6041a03f3214d16c241ca.png"},{"id":75711784,"identity":"b1125082-d824-48e6-8c77-3480c49b86a0","added_by":"auto","created_at":"2025-02-07 11:13:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2623462,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5920650/v1/c7d5b768-1b4d-4379-9454-e586d11aa314.pdf"},{"id":75708748,"identity":"790580d4-6ec2-4199-86b2-06f1f10ee64e","added_by":"auto","created_at":"2025-02-07 10:41:28","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4530350,"visible":true,"origin":"","legend":"Supplementary file","description":"","filename":"SupplementaryfileWBNATURECOMMUNICATIONS.docx","url":"https://assets-eu.researchsquare.com/files/rs-5920650/v1/b4f8163d2bcd2f02d40b1996.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Chronic Suppression of a Multidrug-Resistant Pseudomonas aeruginosa in Prosthetic Joint Infection Using Personalized Bacteriophage Treatment","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMedical implants and devices have transformed medicine by providing a solution to a wide range of conditions that were previously impossible to treat. This has improved quality of life and preserved function while pushing the boundaries in a variety of surgical disciplines including cardiothorasic, vascular, dental, neurological, and orthopaedic fields. About half of all nosocomial infections are associated with medical devices\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. These implant-associated infections are difficult to treat because of their association with antibiotic tolerant biofilm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAn example of this challenge includes total knee and hip arthroplasty (joint replacements). It is one of the most common surgeries in the developed world, and the most severe complication includes periprosthetic joint infections (PJI)\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. It is a devastating diagnosis, often requiring multiple surgical procedures over several years with a five-year mortality between 20 to 25%\u003csup\u003e4,5\u003c/sup\u003e, higher than many cancers\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePeriprosthetic joint infection is difficult to treat because of its association with antibiotic tolerant biofilms. Eradication of the infection requires the removal of components, but surgical intervention with implant removal can have significant morbidity or mortality. Periprosthetic joint infection is associated with multi-drug resistant (MDR) hospital-acquired ESKAPE organisms (\u003cem\u003eEnterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp\u003c/em\u003e, and \u003cem\u003eEscherichia coli\u003c/em\u003e). \u003cem\u003eP. aeruginosa\u003c/em\u003e is a particularly challenging pathogen as conventional antibiotic treatments are often ineffective\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. There is thus an urgent need to develop alternative therapies that can overcome the limitations of antimicrobial-based treatment strategies for implant-associated infections.\u003c/p\u003e \u003cp\u003eLytic bacteriophages, commonly known as phages, have the potential to be an effective therapy against these difficult-to-treat infections because of their innate anti-biofilm activity\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Phage therapy has been used as an adjuvant to antibiotics to treat PJI and other biofilm-associated infections\u003csup\u003e\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. A common strategy includes combining long-term conventional antibiotics with bacteriophage therapy to eradicate or suppress the complex infection.\u003c/p\u003e \u003cp\u003eIn this study, bacteriophage therapy was utilized in a unique way as a stand-alone therapeutic without antibiotics. A patient presented with a chronic multi-drug resistant \u003cem\u003eP. aeruginosa\u003c/em\u003e PJI that was non-operative. They had been referred to palliative care after multiple surgical and antibiotic treatment failures. After standard of care treatment, the patient remained culture positive, but intermittent dosing of bacteriophage resulted in clinical control of the infection. This was achieved, in part, through long-term bacteriophage propagation, limiting biofilm formation, and a phenotypic decrease in bacteria virulence.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eClinical Course and Bacteriophage Therapy\u003c/h2\u003e\n \u003cp\u003eA 96-year-old male with multiple comorbidities including heart failure presented with a chronic multi-drug resistant \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e infection in complex implants. A detailed clinical course and treatment protocol is provided (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea, \u003cstrong\u003eSupplementary information\u003c/strong\u003e). Staged total knee and hip arthroplasties were performed more than 15 years previously that had been complicated by a fracture (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb-e). He was diagnosed with a \u003cem\u003eP. aeruginosa\u003c/em\u003e PJI of his knee that metastasized to his ipsilateral hip (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ef-g). Initially sensitive to oral antibiotics, the organism became multidrug resistant following chronic suppression with ciprofloxacin. Chronic suppression was no longer feasible given the bacteria was resistant to all oral antibiotic options. There were multiple attempts to discontinue intravenous antibiotics over an approximate 2 year period, but this resulted in painful symptomatic infection or sepsis. During each of these recurrences and multiple surgical interventions, the cultures continued to be positive for MDR \u003cem\u003ePseudomonas\u003c/em\u003e.\u003c/p\u003e\n \u003cp\u003eThe patient presented to our institution with a MDR \u003cem\u003ePseudomonas\u003c/em\u003e PJI and complex implants with limited treatment options. At the outside institution, intravenous antibiotics were planned to be stopped permanently with plans for palliative care and hospice. Traditional treatment options were limited including observation with discontinuation of antibiotics, repeat DAIR which had failed the previous four times, removal of components, or amputation. These options had a high probability of leaving him non-ambulatory and with a high expected mortality\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003eGiven limited treatment options, FDA-expanded access was obtained to start bacteriophage therapy. Cultures were obtained and two bacteriophages (PASA16 and \u0026Phi;83)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e (Adaptive Phage Therapeutics (APT), Bethesda, MD) were identified with planktonic and biofilm activity against the \u003cem\u003eP. aeruginosa\u003c/em\u003e clinical isolate tested. Titers of phage for dosing were selected based on maximizing phage titer below the threshold of endotoxin exposure (4.7 x 10\u003csup\u003e10\u003c/sup\u003e plaque-forming units (PFU)/mL PASA16 and 7.5 x 10\u003csup\u003e9\u003c/sup\u003e PFU/mL \u0026Phi;83). Standard of care treatment was provided with debridement and antibiotics with implant retention (DAIR) using intermittent bacteriophage therapy as adjuvant care. The initial dose was intra-articular followed by a daily intravenous dose for one week.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eDiagnostic markers indicated continued infection despite clinical resolution\u003c/h3\u003e\n\u003cp\u003eThe patient was transitioned to chronic suppression with intermittent bacteriophage treatment. Following standard of care treatment, knee aspiration was culture-negative, but continued to meet the Musculoskeletal Infection Society (MSIS) definition of infection\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Plaque assay on the synovial fluid demonstrated the continued presence and propagation of bacteriophage. The patient remained symptomatic with persistent knee effusion and pain at the site for 3 months. Intraarticular bacteriophage dosing was therefore repeated, and the effusion and pain subsided. Based on the initial success and catastrophic consequences of uncontrolled infection, bacteriophage was continued to be dosed at approximately every 4\u0026ndash;6 months without antibiotics. Aspirations completed prior to dosing demonstrated that the patient remained culture positive (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea) and met the International Consensus Meeting (ICM) 2018 definition of prosthetic joint infection\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea-c) but he continued to remain clinically asymptomatic. At 6 months, the knee effusion resolved. This corresponded with an overall decrease in inflammatory markers (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec, \u003cstrong\u003eSupplementary Table\u0026nbsp;1\u003c/strong\u003e). No significant adverse events were observed while on phage therapy. There was a transient elevation in liver enzymes which other groups have previously reported\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed). The patient has remained off antibiotics and has remained asymptomatic at 2-year follow-up with baseline ambulatory function comparable to prior to infection. Long-term clinical control of the infection was established off of antibiotics for more than 2 years. Prior to use of bacteriophage therapy, discontinuation of antibiotics resulted in a painful leg with episodes of sepsis.\u003c/p\u003e\n\u003ch3\u003ePhage lytic activity against patient isolates over time\u003c/h3\u003e\n\u003cp\u003eGiven the extended time period of treatment, phage activity in pre- and post-phage bacterial isolates was examined for phage sensitivity. For analysis, we selected pre-phage (S1 and S2), mid-treatment (S8), and long-term treatment (S22 and S23) \u003cem\u003epseudomonas\u003c/em\u003e isolates. The phages used for treatment, PASA16 (\u003cem\u003ePbunavirus\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea, b) and \u0026Phi;83 (\u003cem\u003ePodoviridae\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec, d), were assessed for activity using standard growth inhibition and a plaque assay \u003cstrong\u003e(Supplementary Table\u0026nbsp;2)\u003c/strong\u003e. For both PASA16 and \u0026Phi;83, growth inhibition assays on pre-phage, mid-term, and long-term phage treatment isolates demonstrated no statistically significant loss of activity (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ee, f). There was a morphological difference between the pre-phage and long-term treatment isolates obtained from the patient. The long-term treatment isolates exhibited a small colony variant phenotype (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;1a, b\u003c/strong\u003e).\u003c/p\u003e\n\u003ch3\u003eNeutralizing antibody production\u003c/h3\u003e\n\u003cp\u003eGiven the multiple doses, the patient\u0026rsquo;s host immunological response to bacteriophage therapy was investigated. Neutralizing antibodies have been observed in serum following intravenous phage therapy in multiple case reports\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. As treatment progressed, we began to monitor for phage-neutralizing antibody activity initially in the serum and then in synovial fluid. Patient specific neutralizing antibodies to these phages were monitored by assessing changes in phage lytic activity when performed in serum or synovial fluid. For treatments involving PASA16, serum collected at 6, 9, 12 and 24 months showed a significant high neutralizing activity and reduced the PFU count (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eg) when compared to the SM\u0026thinsp;+\u0026thinsp;buffer control. Incubation of the phage with patient synovial fluid showed a significant reduction (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.007, Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eh) in PFU count when compared to SM\u0026thinsp;+\u0026thinsp;control. We also compared the patient\u0026rsquo;s serum and synovial fluid against similar samples from an untreated patient. The results were similar, displaying diminished plaque formation in the patient\u0026rsquo;s samples as compared to those from unexposed serum and synovial fluid (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ei, j). Comparable results were observed with \u0026Phi;83 changes in activity from neautralizing antibodies observed in patient serum (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ek) or synovial fluid (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0005, Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003el), but not in unexposed serum and synovial fluid (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003em, n). These findings reaffirm the presence of neutralizing antibodies within both serum and synovial fluid samples, displaying a consistent and marked reduction in viral activity over time when challenged with \u0026Phi;83 and PASA16. This response pattern suggests bacteriophage therapy maintained an active suppression of the infection in the presence of an immunological response from the patient\u0026rsquo;s neutralizing serum and synovial antibodies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBacteriophages reduce and eradicate patient\u003c/strong\u003e \u003cstrong\u003eP. aeruginosa\u003c/strong\u003e \u003cstrong\u003ebiofilm\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eControl of the biofilm associated implant infection suggested the phages used had strong anti-biofilm activity. We hypothesized that bacteriophage therapy could reduce the formation of and potentially eradicate \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilm. We saw a notable impact of bacteriophages \u0026Phi;83 and PASA16 on biofilm formation in \u003cem\u003eP. aeruginosa\u003c/em\u003e clinical isolates collected at different time points. Utilizing both inhibition and eradication assays, concurrent incubation with these bacteriophages led to a significant reduction in biofilm formation compared to non-treated controls. In the inhibition assay, a substantial decrease in biofilm formation was observed. Pre-phage samples S1 and S2 treated with PASA16 and \u0026Phi;83 resulted in a significant decrease in biofilm formation (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0001) compared to untreated controls. Mid-term samples (S8, S13) and late treated isolates (S22, S23) had statistically reduced biofilm formation when treated with PASA16 and \u0026Phi;83 as compared to untreated controls (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0001; Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea\u003cstrong\u003e)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eBiofilms have a high tolerance to antibiotics\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Based on these results, we hypothesized that bacteriophages would have strong anti-biofilm activity. Biofilm was cultured using the clinical isolates, treated with bacteriophage, and remaining biofilm was quantified. Pre (S1, S2), mid-term (S8,13), and late treated (S22,S23) isolates had a significant decrease in biofilm following treatment as compared to untreated controls (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb\u003cstrong\u003e)\u003c/strong\u003e. These results demonstrate a robust capability of both bacteriophages in inhibiting and eradicating biofilms of \u003cem\u003eP. aeruginosa\u003c/em\u003e isolates, and were supported by qualitative observations with Crystal Violet staining. These experiments were repeated with antibiotics. Biofilm were treated with 5x, 20x and 50x MIC of ciprofloxacin (CIP), cefepime (FEP), meropenem (MEM), and piperacillin/tazobactam (P/T). Biofilm remained following treatment (\u003cstrong\u003eSupplementary Fig. 2).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhage therapy reduces\u003c/strong\u003e \u003cstrong\u003eP. aeruginosa\u003c/strong\u003e \u003cstrong\u003evirulence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe hypothesized that bacteriophage treatment may have altered bacteria virulence. The nematode \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e infection model was used as a measure of global virulence (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea). Serial passage was generated with phage growth inhibition assays and was compared with patient isolates for virulence inhibition. \u003cem\u003ePseudomonas\u003c/em\u003e overgrowth from the initial assay was used as the inoculation for a repeat growth inhibition assay. At each repeat passage, phage growth inhibition remained unchanged demonstrating that the bacteria isolate had not developed a phage resistance genotype. \u003cem\u003eC. elegans\u003c/em\u003e survival assays were performed using the patient isolates and \u003cem\u003epseudomonas\u003c/em\u003e isolate S1 from the serial growth inhibition assays using corresponding PASA16 and \u0026Phi;83 (termed S1PASA16\u003csup\u003eOG\u003c/sup\u003e and S1\u0026Phi;83\u003csup\u003eOG\u003c/sup\u003e; Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb, c). \u003cem\u003eC. elegans\u003c/em\u003e infected with serial phage overgrowth \u003cem\u003epseudomonas\u003c/em\u003e (S1PASA16\u003csup\u003eOG\u003c/sup\u003e and S1\u0026Phi;83\u003csup\u003eOG\u003c/sup\u003e) had a comparable survival as \u003cem\u003epseudomonas\u003c/em\u003e S1 that had not been exposed to bacteriophage (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;3a-f)\u003c/strong\u003e. To test other metrics of virulence, swarming (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;4a-f\u003c/strong\u003e) and swimming motility (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;5a-f\u003c/strong\u003e) were measured. Comparing \u003cem\u003epseudomonas\u003c/em\u003e unexposed to phage (S1) to pseudomonas overgrowth from phage (S1PASA16\u003csup\u003eR\u003c/sup\u003e and S1\u0026Phi;83\u003csup\u003eR\u003c/sup\u003e), swarming (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ed, e), swimming (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ef, g), and twitching (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;6a-f\u003c/strong\u003e) was similar as unexposed isolates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenomic analysis of patient\u003c/strong\u003e \u003cstrong\u003eP. aeruginosa\u003c/strong\u003e \u003cstrong\u003eisolates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven the patient\u0026rsquo;s continued positive cultures but overall control of the infection, genomic variation was assessed to quantify any genotype changes in the \u003cem\u003epseudomonas\u003c/em\u003e isolates. Selective pressures from different local environments of the infection can drive adaptive mutations\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Based on pre-and post-treatment with phages and antibiotic susceptibilities patterns (\u003cstrong\u003eSupplementary Table\u0026nbsp;3\u003c/strong\u003e), twenty-seven isolates (pre-phage: S1, S2, S3, S4, S5, S6 and S7; long-term treatment: S8, S9, S10, S11, S12, S13, S14, S15, S16, S17, S18, S19, S20, S21, S22, S23, S24, S25, S26 and S27) were selected for whole genome sequencing (WGS). Each longitudinal isolate was compared with the assembled and annotated genome of the first patient isolate to identify mutations arising during the infection and/or associated with phage therapy (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Nineteen of these isolates were genetically identical to the initial isolate, whereas eight isolates acquired 1\u0026ndash;3 new mutations of unknown relevance to the infection or phage treatment. These findings demonstrate limited evolution of the \u003cem\u003eP. aeruginosa\u003c/em\u003e population during this infection that was controlled by bacteriophage therapy (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). These results also showed that mutations and pseudogenes occurred in pre-mid and post-phage treated samples, but these mutations were not caused by the post-phage treatment and are not consistently present in post-phage treated samples (as shown in \u003cstrong\u003eSupplementary Fig.\u0026nbsp;7a, b\u003c/strong\u003e). WGS data revealed that phages were present on circularized contigs, and the read coverage profile is inconsistent with that of the bacterial genome. Therefore, it indicates that phage genes were present on aspiration but not integrated into the bacterial genome.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cimg 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\"\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAntibiotic resistance is a public health emergency. Multi-drug resistant medical device-related infections illustrate these complex challenges. Here we demonstrate that bacteriophage therapy alone could be used to chronically suppress a multi-drug resistant \u003cem\u003eP. aeruginosa\u003c/em\u003e infection that had failed multiple surgical and antibiotic courses. Clinical control of the infection was established, in part, by bacteriophage modulating bacteria virulence and its activity against biofilm. \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBiofilms are associated with medical device infections and are highly tolerant to antibiotics\u003csup\u003e20,22,23\u003c/sup\u003e\u003csup\u003e3\u003c/sup\u003e as demonstrated by continued treatment failure despite prolonged extended intravenous antibiotics. Bacteriophage has been demonstrated \u003cem\u003ein vitro\u003c/em\u003e to have biofilm activity in other organisms \u003csup\u003e24,25\u003c/sup\u003e, but there is no previous demonstration of possible clinical efficacy. To address this, we tested the efficiency of our bacteriophages on biofilms of the clinical isolates. The results suggest that the bacteriophages reduced and limited \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilm during clinical treatment. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAfter bacteriophage therapy, a phenotypic decrease in \u003cem\u003eP. aeruginosa\u003c/em\u003e virulence was observed. This was observed by a statistically significant increase in \u003cem\u003eC. elegans\u003c/em\u003e survival and reduced motility after long-term phage treatment. A loss of virulence resulted in an ability for the bacteriophage to develop clinical control of the infection without eradication. \u0026nbsp; These changes were not from host adaptation. The pre-phage isolates were an already host-adapted clone that colonized the patient for several years. As well, there was minimal genotype variation between clinical \u003cem\u003ePseudomonas\u003c/em\u003e isolates before and after phage treatment. No phage integration was observed. Host antibodies were observed directed to the bacteriophage, but continued clinical utility continued to be observed. This has been reported in other case reports\u003csup\u003e26,27\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThese findings suggest that the use of bacteriophage therapy resulted in the clinical control of the infection without eradication secondary to phage biofilm activity and a phenotypic alteration in bacteria virulence. This demonstrates an important clinical concept for the ability of bacteriophage to be used in chronic suppression of implant-associated infections. When an antibiotic option does not exist with multidrug-resistant bacteria, our data provides preliminary evidence that bacteriophage may be an alternative therapy. This supports conducting prospective clinical studies.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eEthical statements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe patient has consented to our use of samples and data in research and to the publication of clinical data, the images presented here. He also gave written informed consent to receive phage therapy in accordance with CARE guidelines. For phage therapy, patient cultures were sent to phage company (Adaptive Phage Therapy) and a phage match was obtained. Once phage match was found, we obtained compassionate-use IRB (EA21110067) from the University of Pittsburgh and FDA approval under expanded access (IND 28197).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBacterial strains, bacteriophage and growth conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSynovial fluid and blood samples were collected from the patient during clinical visits. Serum was extracted from blood samples and stored as frozen aliquots at – 80 °C. \u003cem\u003eP. aeruginosa\u003c/em\u003e isolates, collected from the patient’s cultures, were provided by the UPMC Presbyterian Microbiology Lab. Bacteriophages, Φ83 and PASA16, their host cell, C10, and the original patient cultures, S1 and S2, were provided by Adaptive Phage Therapeutics (APT). All bacterial strains were grown in Mueller Hinton Broth (MHB; Bectin Dickinson and Company) medium with shaking at 37 °C and stored as frozen stocks in 30% glycerol at – 80 °C. All phages were tested using a standard plaque assay, expanded, purified and stored as frozen stocks in 30% glycerol at – 80 °C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGrowth inhibition assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS1, S2, S8, S13, S22, and S23 were inoculated into 5 ml MHB medium and grown overnight at 37ºC. Cultures were normalized to 5x10\u003csup\u003e5\u003c/sup\u003e CFU/mL using a 0.5× McFarland turbidity standard (Hardy Diagnostics). The bacteriophages, Φ83 and PASA16, were tittered to approximately 1x10\u003csup\u003e9\u003c/sup\u003e CFU/ml using a standard plaque assay. Cultures were plated in triplicates in 96 well plates (Costar) and bacteriophages were added and incubated at 37 °C for 24 h at shanking incubator. After 24 h the optical density was observed in the microplate reader at 600nm (BioTek).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlaque Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo perform the experiment, 100 μL of each clinical isolate's overnight culture was mixed with 10mL of pre-warmed lysogeny broth (LB) containing 0.75% (w/v) of agar (molten agar) and 5mM of CaCl\u003csub\u003e2\u003c/sub\u003e. This mixture was then poured into 90mm Petri dishes and allowed to solidify. Once the agar had solidified, 10μL drops of PASA16 and Φ83 phage stock, which had been serially diluted ten times, were placed on top of the agar. The drops were left to dry at room temperature and the Petri dishes were then incubated at 37°C overnight. The next day, the plates were checked for lysis zones and the PFU were calculated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMinimum inhibitory concentration (MIC)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCefepime, Ciprofloxacin, Meropenem, and Zosyn minimum inhibitory concentrations for all patient cultures were determined by UPMC Presbyterian Microbiology Lab.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhage-targeting antibody neutralization assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing a standard plaque assay, high-titer lysates of Φ 83 and PASA 16 were created. Patient serum samples from pre-phage and post-phage time points were thawed on ice. Serum samples collected from 3 different timepoints were added to each Φ83 and PASA16 in sterile Eppendorf tubes. SM+ buffer was used as a control. Serum and phage were mixed thoroughly, and then incubated at room temperature overnight. After 24 h, serial dilutions of each mixture were performed in SM+ buffer. Then, 5 uL of each dilution was plated in triplicates onto a top agar lawn of C10. The agar plate was incubated at 37 °C for 24 h. Plaques were counted, and the presence of neutralizing activity was determined in PFU/mL. Additionally, to confirm the presence of neutralizing antibodies this assay was repeated to compare the patient’s serum and synovial fluid samples to control human serum and synovial fluid samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTitanium rod biofilm MIC assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTitanium rods (6 mm) were placed into a 48-well tissue culture plate containing 20 wells of MHB with 1x10\u003csup\u003e6\u0026nbsp;\u003c/sup\u003eCFU/ml P1. The plate was incubated at 37 °C for 48 h. To remove non-adherent cells, wells were replaced with fresh MHB after 24 h. After 48 h, titanium rods were washed in 1 ml PBS and placed into a 48-well tissue culture plate containing Cefepime, Ciprofloxacin, Meropenem, and Zosyn at 1x, 5x, 20x, and 50x MIC. The plate was incubated at 37 °C for 24 h. After 24 h, titanium rods were washed in 1 ml of PBS containing 1% tween 20 (PBST). To break up the biofilm, samples were sonicated using a Branson model #3510 sonicating water bath for 10 min. Serial dilutions of the sonicated bacterial suspension were prepared and plated onto blood agar plates and incubated overnight at 37 °C. Colonies were counted, and the biofilm bacterial burden was determined in CFU/mL. Assay was repeated for S2, S8, S13, S22, and S23.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiofilm inhibition assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS1, S2, S8, S13, S22, and S23 were inoculated into 5 ml MHB medium and grown overnight at 37ºC. Cultures were normalized to 1x10\u003csup\u003e6\u003c/sup\u003e CFU/ml using a 0.5× McFarland turbidity standard (Hardy Diagnostics) and bacteriophages, Φ83 and PASA16, were tittered to approximately 1x10\u003csup\u003e8\u003c/sup\u003e CFU/ml using a standard plaque assay. Phage were added to the cultures to achieve multiplicity of infection (MOI) of 1:100 and plated in triplicates in 96 well plates (Costar) with untreated bacteria as the control. The plate was incubated at 37 °C for 48 h. After incubation, non-adherent cells were removed by turning the plate over and shaking out the liquid. The plate was gently submerged in a small tub of water and then shaken out. Using a micropipette, 200 uL of water were gently added to the wells and then shaken out; this process was repeated 3-4x. Next, 125 uL of crystal violet (CV) was added to the wells and incubated at room temperature for 10-15 minutes. The wells were gently rinsed 3-4x and left to dry upside down in laminar flow hood for 10-15 minutes. To solubilize the CV, 125 uL of 30% acetic acid was added to the wells and incubated at room temperature for 10-15 minutes. To assess biofilm inhibition, absorbance was quantified in a plate reader at 550 nm using 30% acetic acid as the blank. For qualitative purposes, plates were photographed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiofilm eradication assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS1, S2, S8, S13, S22, and S23 were inoculated into 5 mL MHB medium and grown overnight at 37ºC. Cultures were normalized to 1x106 CFU/ml using a 0.5× McFarland turbidity standard (Hardy Diagnostics) and bacteriophages, Φ83 and PASA16, were tittered to approximately 1x10\u003csup\u003e8\u0026nbsp;\u003c/sup\u003eCFU/ml using a standard plaque assay. Cultures were plated in triplicates in 96 well plates (Costar) and incubated at 37 °C for 48 h. After 48 h, Φ83 and PASA16 were added to the wells and leaving 3 wells untreated as the control. The plate was incubated at 37 °C for 28 h. After incubation, non-adherent cells were removed by turning the plate over and shaking out the liquid. The plate was gently submerged in a small tub of water and then shaken out. Using a micropipette, 200 uL of water were gently added to the wells and then shaken out; this process was repeated 3-4x. Next, 125 uL of crystal violet (CV) was added to the wells and incubated at room temperature for 10-15 minutes. The wells were gently rinsed 3-4x and left to dry upside down in laminar flow hood for 10-15 minutes. To solubilize the CV, 125 uL of 30% acetic acid was added to the wells and incubated at room temperature for 10-15 minutes. To assess biofilm eradication, absorbance was quantified in a plate reader at 550 nm using 30% acetic acid as the blank. For qualitative purposes, plates were photographed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSerial passage\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA single colony of the clinical isolate S1 was grown overnight in LB medium on an orbital shaker at 180 rpm for 37°C. Afterward, 100 µL of the overnight culture was diluted in fresh LB medium to maintain a culture of 5 x 10\u003csup\u003e5\u003c/sup\u003e CFU/mL. This culture was then seeded in a 24-well plate, where PASA16 and Φ83 were added to each well, and the phage count was maintained at 5 x 10\u003csup\u003e7\u003c/sup\u003e PFU/mL. The next day, each population was serially propagated by transferring 25 µL of the overnight culture to 1.5 mL of fresh LB medium containing phages. This process was repeated for 10 days. Following the serial passages, we plated the culture on LB plates and counted the CFU the next day. When PASA16 was added to the S1 clinical isolate, the resulting adaptive evolution strain was designated S1PASA16\u003csup\u003eR\u003c/sup\u003e. Similarly, when Φ83 was added to the S1 clinical isolate, the adaptive evolution strain was named S1Φ83\u003csup\u003eR\u003c/sup\u003e. A single colony from these cultures was then picked, allowed to grow in LB medium, and frozen at -80°C in glycerol for future use. This culture was further utilized in experiments to assess \u003cem\u003eC. elegans\u003c/em\u003e survival, as well as swarming and swimming motility.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eC. elegans\u003c/em\u003e maintenance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe wild type \u003cem\u003eC. elegans\u003c/em\u003e (N2 strain) was purchased from Carolina Biological Supply in Burlington, NC, USA for use in this study. The nematodes were grown and propagated on nematode growth media, which consisted of 15g agar, 2.4g NaCl\u003csub\u003e2\u003c/sub\u003e, 2g tryptone, 2.72 g KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 0.8 mL of 1 M CaCl\u003csub\u003e2\u003c/sub\u003e, 5mg/mL of cholesterol, and 1 M MgSO\u003csub\u003e4\u003c/sub\u003e per L. The nematodes were fed with \u003cem\u003eE. coli\u003c/em\u003e OP50 and kept at 25\u003csup\u003eO\u003c/sup\u003eC\u003csup\u003e28\u003c/sup\u003e. To synchronize the worms, 5M NaOH and 5% bleach were used, and eggs were collected. The eggs were washed with M9 medium, which contains 6g Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 3g KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 5g NaCl, and 0.25 g MgSO\u003csub\u003e4\u003c/sub\u003e∙7H\u003csub\u003e2\u003c/sub\u003eO. The eggs were then incubated with S-complete medium (S-basal medium: 5.9 g NaCl, 50 mL of 1M potassium phosphate, pH 6.0 in 1L; S-complete medium: 977 mL S-basal, 10 mL 1M potassium citrate pH 6.0, 10 mL Trace metals solution, 3 ml 1M CaCl\u003csub\u003e2\u003c/sub\u003e, 3 mL 1M MgSO\u003csub\u003e4\u003c/sub\u003e) to allow hatching. For the experiment, age-synchronized L4 worms were incubated in 96-well plates containing 100μL of S-complete medium in each well.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eC. elegans\u003c/em\u003e\u003c/strong\u003e \u003cstrong\u003e\u003cem\u003ein vivo\u003c/em\u003e infection model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the infection assay, 96-well plate was filled with S-complete medium added with overnight culture of \u003cem\u003eE. coli\u003c/em\u003e OP50. Then 15 mature L4 stage \u003cem\u003eC. elegans\u003c/em\u003e were transferred in to medium. The total volume in the 96 well plate was maintained at 100 μL. Next, clinical isolates (\u003cem\u003eP. aeruginosa\u003c/em\u003e) were grown overnight and washed with PBS and maintain the 1x10\u003csup\u003e6\u003c/sup\u003e CFU/mL for infection. The efficacy of phage therapy was studies using concentration of bacteria and phage with 1:100 multiplicity of infection (MOI). The Group 1 (control) consisted of S-complete medium with 15 nematodes, no bacteria. Group 2 (vehicle control): S-complete medium with 15 nematodes, no bacteria, PBS. Group 3 (infection control): S-complete medium with 15 nematodes infected with S1. \u0026nbsp; Group 4 (phage treatment): S-complete medium with 15 nematodes infected with S1 and treated with PASA16. Group 5 (phage treatment): S-complete medium with 15 nematodes infected with S1 and treated with Φ83. Group 6 (infection control): S-complete medium with 15 nematodes infected with S2. \u0026nbsp;Group 7 (phage treatment): S-complete medium with 15 nematodes infected with S2 and treated with PASA16. Group 8 (phage treatment): S-complete medium with 15 nematodes infected with S2 and treated with Φ83. Group 9 (infection control): S-complete medium with 15 nematodes infected with S3. \u0026nbsp;Group 10 (phage treatment): S-complete medium with 15 nematodes infected with S3 and treated with PASA16. Group 11 (phage treatment): S-complete medium with 15 nematodes infected with S3 and treated with Φ83. Group 12 (infection control): S-complete medium with 15 nematodes infected with S4. \u0026nbsp; Group 13 (phage treatment): S-complete medium with 15 nematodes infected with S4 and treated with PASA16. Group 14 (phage treatment): S-complete medium with 15 nematodes infected with S4 and treated with Φ83. Group 15 (infection control): S-complete medium with 15 nematodes infected with S5. \u0026nbsp;Group 16 (phage treatment): S-complete medium with 15 nematodes infected with S5 and treated with PASA16. Group 17 (phage treatment): S-complete medium with 15 nematodes infected with S5 and treated with Φ83. Group 18 (infection control): S-complete medium with 15 nematodes infected with S6. \u0026nbsp;Group 19 (phage treatment): S-complete medium with 15 nematodes infected with S6 and treated with PASA16. Group 20 (phage treatment): S-complete medium with 15 nematodes infected with S and treated with Φ83. The plates were incubated at 20\u003csup\u003eO\u003c/sup\u003eC, and survival was monitored every 24 h for 7 days. The results were observed and evaluated based on live and dead \u003cem\u003eC. elegans\u003c/em\u003e. All the experiments were repeated a minimum of three times for statistical significance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMotility assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSwarming Motility Assay\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSwarming motility was tested as reported previously with a few modifications\u003csup\u003e29\u003c/sup\u003e. To create the swimming plates, 6.5 g of agar was added to 800 mL of water, and the mixture was then autoclaved. 200 mL of a 5× M8 solution was then added to the agar mixture. The 5× M8 solution was created in advance by combining 30 g Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 15 g KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, and 2.5 g of NaCl with enough water to obtain a final volume of 1 L. After the addition of the 5× M8 solution, 10 mL of 20% glucose, 25 mL of 20% casamino acids, and 1 mL of 1 M MgSO\u003csub\u003e4\u003c/sub\u003e were combined with the agar solution. The solution was mixed and cooled prior to being poured in petri dishes (〜25 mL/plate), and the plates were allowed to solidify overnight at room temperature. For the experimental test plates, 10 μL of an overnight bacterial culture from a clinical isolate was combined with 10 μL of the phage being tested (PASA16 or ɸ83). This step was repeated for each of the 6 clinical samples being tested. The plates were then inoculated with 2.5 μL of bacteria, or 2.5 μL of the bacteria and phage mixture, and incubated upright for 24 hours. Following incubation, the zone diameter was recorded for each plate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSwimming Motility Assay\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSwimming motility was analyzed as previously described with minor modifications\u003csup\u003e29\u003c/sup\u003e. Plates were created as previously described for the Swarming Motility Assay, but 3 g of agar was used. For the experimental test plates, 10 μL of an overnight bacterial culture from a clinical isolate was combined with 10 μL of the phage being tested (PASA16 or ɸ83). A toothpick was dipped into this mixture, and stabbed partway into the agar plates. The plates were then incubated upright for 24 hours, and the zone diameter was measured.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTwitching Motility Assay\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwitching motility was accessed as previously described with minor \u0026nbsp;modifications\u003csup\u003e30\u003c/sup\u003e. LB agar plates were stab inoculated with a mixture of 10 μL of an overnight bacterial culture from clinical samples and 10 μL of the phage being tested (PASA16 or ɸ83). The plates were then incubated for 48 hours and the agar was removed. The remaining attached cells were stained with a 1% Crystal violet solution, and the size of twitching was then measured.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll graphical and statistical analysis was performed using Prism 9.5 (GraphPad, La Jolla, CA). Statistical analysis was performed using one-way ANOVA with multiple comparisons test for the neutralizing antibodies data, and an unpaired t-test for the biofilm data. Significance was determined at p \u0026lt; 0.05.\u0026nbsp;Images were treated with ImageJ (v1.53).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWhole-genome sequencing and Raw read processing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDNA from twenty-seven \u003cem\u003eP. aeruginosa\u003c/em\u003e isolates was extracted using Qiagen kit according to the manufacturer’s protocol. Briefly, genomic DNA was isolated from frozen cell pellets using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) specifically tailored for the QIAcube. The elution process was conducted over a 10-minute period into nuclease-free water to ensure the purity of the DNA. The concentration of the extracted DNA was determined using the Qubit® 3.0 Fluorometer. Later, sample was sent to SeqCoast Genomics for further analysis.\u0026nbsp;Trimmomatic (version 0.39) was used with the following parameters to quality-trim reads based on phred33 quality scores and remove adaptor sequences\u003csup\u003e31\u003c/sup\u003e. Genomes were assembled using the Unicycler software (version 0.4.4)\u003csup\u003e32\u003c/sup\u003e. Annotation was performed using BAKTA (version 1.6.1)\u003csup\u003e33\u003c/sup\u003e. Comparative genomics was performed using DIAMOND (version 2.0.15.153, Buchfink \u003cem\u003eet al\u003c/em\u003e., 2015) and the Pseudofinder software\u003csup\u003e34\u003c/sup\u003e. Variant-calling was performed using the Breseq software (version 0.36)\u003csup\u003e35\u003c/sup\u003e. Breseq was run with the consensus-minimum-variant-coverage-each-strand flag set to 3, and all other default parameters. Sample 1 was used as the reference genome, and false-positive mutations were filtered out based on their presence in sample 1. Principal component analysis was performed using the R package \u003cem\u003efactoextra.\u003c/em\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work would not have been possible without the support of Robert Hopkins, MD, Kendra Weaver, Acknowledgements Reddy, \u0026nbsp; \u0026nbsp;Anantha Makineni, Aravinda Vadlamudi, Jordan Erickson, Laura Hauns. \u0026nbsp;The funders had no role in shaping the conceptual framework, designing the study, collecting or analyzing data, deciding to publish, or preparing the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the data analysis and outlined the study; R.A. and K.L.U designed\u003c/p\u003e\n\u003cp\u003ethe experiments. R.A., N.M.S., A.J.F. and J.J.R. conducted most of the studies; V.S.C. conducted the bioinformatic analysis and analysed the data; R.A., K.L.U wrote the manuscript with contributions from other authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrespondence and requests for materials\u003c/strong\u003e should be addressed to Kenneth L. Urish\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDarouiche, R.O.J.N.E.J.o.M. Treatment of infections associated with surgical implants. \u003cstrong\u003e350\u003c/strong\u003e, 1422-1429 (2004).\u003c/li\u003e\n\u003cli\u003eHall-Stoodley, L., Costerton, J.W. \u0026amp; Stoodley, P.J.N.r.m. 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Infections associated with medical implants serve as an example. Conventional antibiotics have limited ability to eradicate these infections as they are associated with antibiotic-tolerant biofilms. Here, we report the use of bacteriophage therapy for the treatment of a MDR, non-operable \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e periprosthetic joint infection that had failed multiple antibiotic and surgical interventions. Treatment with intermittent bacteriophage therapy alone without antibiotics over a 2 year time period resulted in clinical resolution of the infection, but not microbiological eradication. Bacteriophage therapy established this control, in part, by altering bacterial virulence and disrupting biofilm. Whole genome sequencing demonstrated the continued presence of bacteriophage during treatment without integration into the bacteria genome. This provides preliminary evidence that bacteriophage can be used to treat multidrug-resistant infections when antibiotic options do not exist.\u003c/p\u003e","manuscriptTitle":"Chronic Suppression of a Multidrug-Resistant Pseudomonas aeruginosa in Prosthetic Joint Infection Using Personalized Bacteriophage Treatment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-07 10:41:23","doi":"10.21203/rs.3.rs-5920650/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"303b90a2-0842-486d-b655-710e630a4179","owner":[],"postedDate":"February 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":43934485,"name":"Biological sciences/Microbiology/Bacteriophages"},{"id":43934486,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2026-04-09T15:31:15+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-07 10:41:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5920650","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5920650","identity":"rs-5920650","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}