Timely bespoke phage-antibiotic combination to treat refractory Pseudomonas aeruginosa mediastinitis and vascular graft infection

Domain: biology
Published: 2026-01-09
Editorial outcome: minor_revision

Abstract
Biofilm-related vascular graft infections (VGIs) pose major therapeutic challenges due to persistent, antibiotic-resistant bacteria often residing in retained grafts. Phage therapy offers a promising alternative treatment strategy against biofilm-associated infections, though its user remains mostly ad hoc and typically considered a last-resort intervention. We report here the treatment of a refractory, fluoroquinolone non-susceptible Pseudomonas aeruginosa VGI using a systematically planned and synergistic phage-antibiotic combination approach. Adjunctive phage therapy led to radiological improvement, as seen by reduced 18F-FDG PET/CT tracer uptake around the graft. The patient was transitioned to oral fluoroquinolone suppression therapy with no recurrence of bacteremia to-date, after a year. Our workflow led to the selection of phages that sensitized Pseudomonas aeruginosa to killing by levofloxacin and piperacillin-tazobactam. We established that this phage-driven antibiotic sensitization was due to the ability of our phages to use the MexAB-OprM efflux pump as a receptor. We also showed that our phages had potent anti-biofilm activity. We advocate a systematic, multi-pronged management strategy for refractory VGIs, including early therapeutic drug monitoring (TDM), in vitro antibiotic combination testing (iACT), and timely adjunctive phage therapy. This case illustrates the utility of individualized, strategic approaches and highlights adjunctive phage therapy’s potential in treating complex biofilm-related infections.

Introduction
Vascular graft infections (VGIs), although uncommon, are serious infections following arterial reconstruction surgeries. VGIs are associated with significant morbidity and mortality, especially in patients with intra-cavity VGIs1–3. Definitive treatment with graft explant is not always possible due to patient factors and surgical technical challenges, especially in patients with composite valve grafts (e.g., ascending aortic graft infections)3. If the graft is preserved, treatment of the VGI with a prolonged course of parenteral antibiotics followed by long-term suppressive antimicrobial therapy is often recommended due to the presence of biofilm; survival rates are low and infection recurrences common4,5. In refractory VGIs, the infected grafts are often left in situ; the causative bacteria often form drug-tolerant biofilm, and the emergence of persistent bacteria subpopulations is not unexpected6,7. With time and ongoing antibiotic selection pressure, VGIs are likely to become drug resistant, and harder to treat.

The development and early application of biofilm-targeting novel therapeutic strategy is crucial for the treatment of refractory VGIs; adjunctive bacteriophage (phage) therapy serves this purpose, and interest in this field is growing8–10. Phages are viruses that specifically infect bacterial cells and can cause bacterial lysis. Using phages for treatment offers several advantages9. Phages have been shown to be active against both planktonic and biofilm-forming bacteria. Phages have also been shown to enhance antibiotic activity by eliciting phage-antibiotic synergies and restoring susceptibilities of otherwise antibiotic-resistant bacteria to antibiotics10. In a targeted review exploring the role of phages (used in combination with antibiotics) in VGIs, bacterial eradication was achieved in 6 out of 7 (86.6%) cases where either Staphylococcus aureus and/or Pseudomonas aeruginosa (P. aeruginosa) were isolated, both notorious for biofilm formation. Clinical improvement was seen in 4 out of 7 (57.1%) cases9.

A 36-year-old female patient with a history of congenital heart disease (pulmonary atresia with ventricular septal defect) with 4 prior sternotomies for corrective heart surgery developed progressive dilatation of her ascending aorta with severe aortic regurgitation and pulmonary homograft stenosis in 2023. This was associated with admissions for decompensated heart failure. To address this, she eventually underwent a Bentall procedure where the affected aortic valve, aortic root and ascending aorta were replaced by a composite vascular graft and the pulmonary homograft was also replaced in the same setting on January 25th, 2024, just 5.5 weeks after she had delivered her second child. Surgery was technically challenging due to dense adhesions from prior sternotomies with significant intra-operative bleeding, and inadvertent tear in the aorta during adhesiolysis for which gel weave patch augmentation of the inferior aspect of the aortic arch was performed.

Her post-operative course was eventful and protracted (See Fig. 1). She developed a febrile illness on post-operative day (POD) 2 and was treated for culture-negative healthcare associated infection with a 5-day course of piperacillin-tazobactam with response. However, on POD 9, she developed drug sensitive P. aeruginosa mediastinitis. (See Fig. 2A.) Mediastinal washout performed on POD 15 showed good clinical and biochemical response. When her condition stabilized, she was transitioned to oral ciprofloxacin 750 mg, completing 8 weeks of antibiotics, with normalization of white blood cell (WBC) 8.32×109/L and C-reactive protein (CRP) was 11.2 mg/L at end of therapy.

However, within 4 days of discontinuing antibiotics, her fever recurred. She only presented to care 10 days later with ongoing fever and chills with an elevated, WBC 16.71×109/L, CRP 283 mg/L and procalcitonin 12.4 μg/L. She also suffered a high-grade P. aeruginosa bacteremia which had evolved to be ciprofloxacin non-susceptible. Although the CT scan showed no signs of recurrent mediastinal abscess, metastatic infection, or infective endocarditis, there were significant concerns about the progression of refractory or resistant intra-cavity thoracic VGIs. Due to the high surgical risk, a redo Bentall procedure was considered unsafe.

In view of the above, a multi-disciplinary team was convened, and we opted for a systematic and individualized approach to manage the P. aeruginosa VGI. Tailored antibiotic therapy, guided by therapeutic drug monitoring (TDM) and in vitro antibiotic combination testing (iACT), was implemented. Additionally, personalized phage therapy was considered early and integrated into the treatment plan. This resulted in the timely administration of salvage phage therapy and restoration of oral fluoroquinolone efficacy supporting chronic suppression of the VGI.

Results
Systematic approach – Optimizing antibiotic regimen and initiating phage workflow
In mid-April 2024, the patient was reinitiated on treatment for recurrent P. aeruginosa bacteremia. Biomarker response to the piperacillin-tazobactam 4.5 g q6h ly, breakthrough fever and recurrent bacteremia occurred quickly within 5 weeks of therapy. In response, TDM was performed to guide antibiotic dosing and improve care (Table S1). Antibiotics dosages were adjusted based on attainable drug levels for a deep-seated infection, biofilm activity, “perceived” or proven synergism in vitro, as well as feasibility of outpatient antibiotic administration. (Fig. 1. Details are described in the Supplementary Information).

With standard and even high dose drug administration with prolonged infusion, the serial β-lactam drug levels were deemed inadequate for the treatment of deep tissue infection(s) (detailed Supplementary Information). It was therefore not surprising that recurrent breakthrough of bacteremia occurred (three episodes in 3 months). The patient was subsequently placed on high-dose piperacillin-tazobactam 27 g/day and oral levofloxacin 750 mg daily in June 2024 based on synergy E-test results and the bacteremia cleared. (See the Supplementary Information.)

Given the refractoriness of the VGI, a multi-disciplinary team comprising infectious diseases physicians, microbiologists, pharmacists, treating cardiothoracic surgeons, and phage researchers was convened in June 2024, and consensus recommendations for compassionate use of adjunctive phage therapy was made. Thereafter, a collaborative effort within the Singapore Phage Network was initiated. The decision to pursue adjunctive salvage phage therapy was driven by her clinical course and serial 18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) findings, which showed persistent tracer uptake in the aortic graft despite prolonged high-dose combination antibiotic therapy guided by TDM. However, the risk of relapse once antibiotics were discontinued was concerning. (See Fig. 2.) In addition, there were interval development of vascular access complications: Vascular catheter exit site infection developed on September 4, 2024, necessitating line replacement; long term intravenous antibiotic therapy was not tenable. Definitive surgery to remove the composite vascular graft was technically challenging with high operative risks, significant morbidity and mortality, which was unacceptable to the patient. Phage therapy offered the possibility of restoring antibiotic susceptibility to fluoroquinolones and beta-lactams with the aim to suppress the VGI. Approval for the use of investigational phage therapy was obtained from our hospital medical ethics committee. The patient was also counselled, and she later consented to receiving phage-antibiotic combination therapy as part of salvage treatment.

Microbiological profiles
With the first recurrence of P. aeruginosa bacteremia in April 2024, the bacterial isolates from this patient were sequentially archived and used to screen our phage repository, as well as to evaluate phage-antibiotic combination, and to produce selected phages for compounding of the selected therapeutic cocktail. A total of 4 blood culture P. aeruginosa isolates (PA2081, PA2091, PA2096, and PA2111) were retrieved between April and June 2024. Despite morphological differences, they exhibited similar antibiotic susceptibility profiles (see Tables S2, S3). All four isolates belonged to ST252 and shared the similar resistance/virulence gene profiles (Table S4 in the Supplementary Information). Pairwise genomic alignment has reflected a highly conserved profile with genomic similarity between 98.3% to 99.8% across isolates. The prophages (or Phage Defense System) in these 4 isolates were identified and characterized (Table S4 and Fig. S1). Prophages are devoid of VF, AMR genes or other remarkable genetic elements.

Identification of suitable therapeutic phages
The Singapore Phage Repository—established through collaboration between Singapore General Hospital, Singapore Eye Research Institute, National University of Singapore, and Nanyang Technological University—was systematically assembled to include therapeutic-grade phages with potent lytic activity against clinically relevant pathogens. Phages were isolated using well-characterized clinical isolates of P. aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, and Staphylococcus aureus, representing prevalent sequence types and resistance profiles from Singapore General Hospital and other local institutions. All phages underwent whole-genome sequencing (WGS) and annotation to confirm their lytic nature and exclude those carrying antimicrobial resistance genes, virulence factors, or lysogeny-associated markers. Only strictly lytic, genomically “clean” phages were retained, ensuring a diverse, well-characterized collection suitable for precision phage therapy. This repository served as the primary phage source for this case.

Identification of Phage Candidates and Selection of Therapeutic Phage Cocktail
A multidimensional evaluation workflow (Fig. 3) was used to identify the most suitable therapeutic phages from the Singapore Phage Repository. Screening began with phage susceptibility testing of the four P. aeruginosa clinical isolates using spot and plaque assays. Eight distinct phages demonstrated infectivity (Figs. S2, S3). Lytic activity was quantified through time-kill assays, assessing initial bacterial killing, regrowth kinetics, and final bacterial counts at 24 h. Five single phages, PW20, PW21, PW23, KSY1a, and P0413, were shortlisted based on diverse killing profiles and selection pressures (Figs. S4–S7).

From these candidates, we evaluated 20 phage–phage combinations (two- and three-phage cocktails), identifying potential phage-phage synergistic activities. Four 3-phage cocktails exhibited superior performance, achieving greater initial killing, sustained suppression of bacterial regrowth, and delayed rebound compared with single phages or 2-phage cocktails. However, none achieved complete eradication without antibiotic co-administration (Figs. S8, S9).

Phage Cocktail #3, comprising PW21, KSY1a, and P0413, was selected for therapeutic use as it demonstrated consistent high potency against both planktonic and biofilm cultures of all four P. aeruginosa clinical isolates, robust synergistic activity with key antibiotics (e.g., tazobactam, levofloxacin) (Fig. 4A–D, Table S9, and Figs. S10–S16), and broad coverage with minimal cross-resistance (Table S10). In addition, all three constituent phages were genomically confirmed as strictly lytic and free of undesirable genes and remained available in high-titer stocks suitable for intravenous administration.

Phage-Driven Antibiotic Sensitization
Phage-driven antibiotic sensitization was identified by assessing minimum inhibitory concentration (MIC) shifts and enhanced bacterial killing in combination assays. MIC testing of the four patient isolates revealed substantial reductions when antibiotics were combined with Phage Cocktail #3, often restoring susceptibility (Fig. 4A–C). Kill-kinetic growth inhibition assays demonstrated that the combination of cocktail #3 (individual phages or as a cocktail) and piperacillin–tazobactam or levofloxacin, achieved sustained killing below the limit of detection at MIC concentrations and enhanced inhibition at sub-MIC levels (Fig. 4A–D). Viability and biofilm biomass assays further confirmed that phage cocktails not only dispersed mature biofilms but also ensured bacterial killing (Fig. 4E). We also established that PW20, PW21 and PW23 rely on MexAB-OprM membrane proteins to infect P. aeruginosa. The ability of PWs phages to utilize the MexAB-OprM efflux pump as a receptor provide the foundation for the underlying mechanism of phage-driven antibiotic sensitization (Fig. S17). The genomic profiles of the three therapeutic phages selected (PW21, KSY1A, and P0413) are provided in the Supplementary Information (Tables S5–S8). PW21 and P0413 (both Myoviridae) have genome lengths of 67,991 bp and 66,492 bp, respectively, while KSY1a (Phikzvirus) has a significantly larger genome of 282,489 bp, which makes it a jumbo phage.

Intravenous phage treatment
Phage therapy administration and monitoring was adapted from the STAMP protocol11. A 2-week course of purified 3-phage cocktail, PW21, P0413 and KSY1a were selected, and phage titers between 108 and 109 plaque-forming unit (PFU) was finally administered (daily on the first 2 days, and twice daily for the remaining 12 days) in combination with her existing antibiotic regimen (piperacillin-tazobactam and levofloxacin) on September 23, 2024.

Clinical course following phage therapy
The patient tolerated phage therapy well with no adverse events observed during treatment. Biochemically, we noted a measurable increase in the inflammatory markers; the previously normal CRP and erythrocyte sedimentation rate (ESR) increased to a peak of 16.8 mg/L, and 44 mm/h, respectively, after phage administration.

The patient was symptom free before and after phage therapy with no impairment in daily activities. The most significant observation following phage administration was the reduction in tracer uptake around the prosthetic aortic valve 1 and 2 months after phage therapy on serial 18F-FDG PET/CT (Fig. 2F). The serial CRP and ESR readings also returned to normal limits 15 days, and 85 days post phage therapy, respectively. Intravenous piperacillin-tazobactam was discontinued on December 3, 2024, and the central venous catheter was removed. The patient was subsequently maintained on levofloxacin monotherapy with no recurrence of bacteremia.

Incorporating systematic therapeutic monitoring during phage therapy
Here, we performed therapeutic phage monitoring, which is clinical monitoring and evaluation of patients while undergoing phage therapy, to 1) assess safety profile, specifically to identify unwarranted, adverse immune reactions triggered by phage administration; 2) detect phages in patients. Phages are nucleoprotein complexes which are potentially highly immunogenic, particularly when administered intravenously. Clinically, the patient exhibited no signs or symptoms indicative of systemic inflammation or anaphylactic responses. However, slight increases in CRP and ESR, as previously noted, alluded to a mild inflammatory response post phage administration. Phage were detected in patient blood samples at day 8 and day 15 of phage therapy by PCR using primers designed to discriminate between the three phages (Figs. S18, S19 and Table S11).

Methods
Ethical statement
Informed consent for the compassionate use of phage therapy was obtained from the patient and prior approval to proceed was sought from the SingHealth Translation Medicine Office. According to the CARE guidelines and in compliance with the Declaration of Helsinki principles, the therapeutic monitoring and clinical outcome assessment of adjunctive phage therapy was approved by the SingHealth Centralized Institutional Review Board (CIRB 2023/2415). Written informed consent for publication of clinical details and clinical images was obtained from the patient. A copy of the consent form is available for review by the Editor of this journal.

Sample and isolate collection
Bacterial isolates were recovered from routine clinical cultures of the patient blood. P. aeruginosa isolates were sequentially collected before phage therapy. All isolates were identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (Bruker Daltonics, Billerica, MA, United States) using in-vitro diagnostic (IVD) library revision K (2020) software following the manufacturer’s instructions. Isolates were stored in 20% (v/v) glycerol at −80 °C.

Phage susceptibility testing via spot test
Double agar overlay assay was used to determine the susceptibility of isolates to phages. Briefly, phage stocks were serially diluted in SM buffer containing 50 mM Tris-Cl pH 7.5 (Axil Scientific, Singapore), 100 mM NaCl (Sigma Aldrich, MO, United States), 8 mM MgCl2 • 7H2O (VWR), and spotted on bacterial lawn. Agar plates were left to incubate overnight at 35 °C.

Microtiter MIC assays
Overnight bacterial cultures were adjusted to logarithmic growth phase (until OD630 ~0.08) and further diluted in fresh Mueller Hinton II Broth (Cation-Adjusted) (Becton-Dickinson [BD] BBLTM, NJ, United States) to obtain an approximate bacterial inoculum of 105.7 colony forming units (CFU)/mL. Serial dilutions of phage were then added to the bacterial culture to achieve different multiplicities of infection. Bacteria-phage mixture was incubated for 20 min at 35 °C. A 100 μL of this mixture was added to each well of the customized 96-well microbroth dilution panels (TREK Diagnostics, OH, United States) lined with antibiotics, in accordance with the manufacturer’s recommendations and incubated for 16–20 h at 35 °C. Susceptibility and breakpoints were interpreted according to Clinical & Laboratory Standards Institute36. P. aeruginosa ATCC 27853 was used as the quality control strain. MIC value is the lowest concentration of an antibiotic at which bacterial growth is completely inhibited, as indicated by clear (non-cloudy) well.

Time-kill assay
Overnight bacterial cultures were adjusted to logarithmic growth phase (until OD630 ~0.08) and further diluted in fresh Mueller Hinton II Broth (Cation-Adjusted) (Becton-Dickinson [BD] BBLTM, NJ, United States) to obtain an approximate bacterial inoculum of 106 CFU/mL. Bacteria were either infected with phage(s) at a multiplicity-of-infection of 100 or added with antibiotics at their achievable unbound body concentrations. Antibiotics added are aztreonam (0.5 mg/L), ceftazidime/avibactam (0.5/4 mg/L), piperacillin/tazobactam (4/4 mg/L), fosfomycin (16 mg/L), levofloxacin (0.5 mg/L), ceftolozane/tazobactam (0.5/4 mg/L) and meropenem (0.5 mg/L). Each antibiotic was used at its MIC level as determined in the microtiter MIC study with the four-bacteria mixture and the three-phage cocktail (Fig. 3C). For samples treated with phage-antibiotics combinations, bacteria were first infected with phage and incubated at 35 °C for 20 min before exposing to antibiotics. All samples were incubated for 24 h at 35 °C in a shaking incubator. At different time points (2, 4, 6, 8 and 24 h), a small aliquot (1 mL) was sampled and plated on Müeller Hinton agar plates for colony enumeration. Synergism between 2 treatment modalities is defined as at least 2 log10 CFU per ml reduction from the most active single treatment modality at 24 h37–39. Additivity is defined as 1 to less than 2 log10 CFU per ml reduction from the most active single treatment modality at 24 h. Bactericidal activity is exhibited as 3 log10 CFU per ml reduction from the baseline (at time 0 h) inoculum. Neutrality is defined as less than 1 log10 CFU per ml reduction from the most active single treatment modality at 24 h. Antagonism is defined as at least 2 log10 increase in CFU/mL at 24 h for the combination compared to the most active single modality.

Biofilm dispersion assay
We tested the ability of our phage cocktails to disperse biofilm in vitro using crystal violet (CV) staining as a proxy for biofilm biomass, and CFU enumeration as a measure of bacteria viability. Our crystal violet staining protocol is derived from the original work of Christensen et al.40. Briefly, P. aeruginosa (patient’s isolates) were pre-cultured to exponentially growing phase in LB. 1 mL of each culture was pelleted at 8000 rpm for 3 min, washed twice in M63 media and resuspended to a OD600 of 0.6. This resuspension was used to inoculate 96-well plate (190 μL of M63 + 10 μL of resuspension). Plates were incubated for 6 h or 24 h, and biofilm was allowed to form on the well’s surface before being washed carefully with sodium-magnesium (SM) buffer (50 mM Tris-HCl [pH 7.5], 100 mM NaCl, 10 mM MgSO4) before phage cocktails in SM buffer were added for an additional 24 h. For CV staining and biofilm biomass quantification, supernatant was carefully discarded after 24 h of treatment, wells were washed thrice with MiliQ water, and plates allowed to dry for 1 h. Next, 200 μL of 0.1% crystal violet solution (5% methanol, 5% isopropanol) in MiliQ water was added and plates were rocked for 30 min before rinsing three times and dried for 2 h at room temperature. Finally, 200 μL 20% acetic acid in MiliQ water was added to solubilize the CV before OD595 was measured using a plate reader. To determine viability of bacteria within the biofilm, the supernatant was carefully discarded after 24 h of treatment, wells were washed thrice with M63 media and the remaining biofilm was resuspended in LB. Serial dilutions were plated on LB plates and incubated for 24 h before CFU enumeration.

Phage preparation
Phages PW21, KSY1a and P0413 were generated and purified according to Pirnay et al.22, with minor modifications. Briefly, phages were co-cultured with bacteria host strain PA2111 in 4 LB BBLTM Müeller Hinton II Broth, Cation adjusted, at 35 °C for 16 h. Lysate was filtered through 0.22 μm filter to sterilize, pelleted by centrifugation at 10,000 xg for 90 min at 4 °C and resuspended with Infusol® NS 0.9% (w/v) saline. Endotoxins were depleted by passing the sample through Lionex EndoTrap® HD 1 mL column three times. Endotoxin purified phage suspensions were filtered using medical grade 0.22 μm filter, collected and aliquoted into sterile, depyrogenated 2.0 mL glass vials. Sterility and bacterial endotoxins testing were performed, according to USP 71 and USP 85 standards respectively, by Setsco Services Pte Ltd (Singapore). A phage product meets the endotoxin safety threshold for intravenous use if it can be administered at <5 EU/kg/h.

Phage genomic DNA extraction and whole genome sequencing
Genomic DNA from pure phage preparation was extracted using Norgen Phage DNA Isolation Kit (Norgen Biotek, Thorold, ON, Canada) according to the manufacturer’s guidelines. DNA concentration was measured using the Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). Raw reads, from NovoSeq systems (Illumina Inc., CA, USA), were trimmed and assembled with an in-house pipeline using Trim Galore (0.6.10) and SPAdes (3.15.5), respectively. Host contamination was identified with bwa-mem2 and removed with in-house bash code using bwa-mem2 and reassembled with SPAdes again. Assembled contigs were then annotated with pharokke (1.7.0). Taxonomy was identified according to the closest match in database with KmerFinder and BLASTn (v2.13.0) against nr. Phage lifestyle was then predicted with PhageAl and the presence/absence of integrase. Assembled contigs of phages were analyzed with ResFinder and CARD RGI to identify AMR genes and were BLASTn against the VFDB (2022-Sep-23 update) to confirm the absence of virulence genes.

Determination of Phage-driven antibiotic sensitization
Briefly, growth inhibition assays were carried out in 96-well plates in presence of increasing concentration of either levofloxacin or piperacillin/tazobactam, combined with either phages alone or combined in a cocktail. OD600 was measured after 24 h of incubation and reported as a function of antibiotic concentration. MIC shift was inferred from these results. Phage-driven antibiotic killing at sub-MIC concentration was determined by treating an inoculum of 106 bacteria with either a sub-MIC concentration of levofloxacin or piperacillin/tazobactam, alone or in combination with phages. Viability was determined after 24 h incubation by CFU enumeration.

Amplification of Phage DNA from Patient Blood Samples
Described in Supplementary Information
Detailed methods and results for TDM, Synergy E-test, antibiotic susceptibility profile of the P. aeruginosa isolates, transmission electron microscopy imaging of the phages, and phage immune monitoring are available in Supplementary Information.

Statistics & Reproducibility
No statistical method was used to predetermine sample size. No data were excluded from the analyses. The experiments were not randomized; The investigators were not blinded to allocation during experiments and outcome assessment. Line graphs and scatter plots were generated using GraphPad Prism version 10.0.0 for Windows, GraphPad Software, Boston, Massachusetts USA.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This case report describes the use of personalized phage and antibiotic combination therapy to manage a refractory\nPseudomonas aeruginosa vascular graft infection. The patient experienced multiple episodes of recurrent bacteremia\ndespite extended courses of high dose antibiotics, including piperacillin tazobactam and fluoroquinolones. Phage therapy\nwas pursued due to the persistent biofilm nature of the infection, the emergence of fluoroquinolone resistance, and the\nelevated surgical risk that ruled out graft removal.\nA three-phage cocktail was selected based on its in vitro lytic activity and biofilm disruption capacity. It included two closely\nrelated Pbunavirus phages (PW21 and P0413) and a jumbo PhiKzvirus phage (KSY1a). Phage therapy was administered\nIV for two weeks alongside ongoing treatment with piperacillin tazobactam and levofloxacin. At the time of phage initiation,\nblood cultures were negative, indicating resolution of bacteremia, but 18F FDG PET/CT imaging continued to show\nmetabolically active infection.\nThe addition of phage therapy was considered relatively safe. The patient experienced no clinical adverse events and\nremained well (line 281), despite a transient but measurable rise in inflammatory markers including CRP and ESR. Although\nthese returned to baseline after treatment (lines 283 to 289). Imaging on October 24 showed radiologic improvement, and the\npatient was transitioned to oral fluoroquinolone suppression therapy.\nThe study contains several strengths, including a multidisciplinary treatment approach, early integration of phage therapy\ninto the care plan, rigorous screening of bacterial isolates, and therapeutic drug monitoring. However, it also has important\nlimitations. The main concern is that the evidence supporting phage efficacy is circumstantial and mechanistically plausible\nbut not definitive. The patient was already showing improvement on the September 10 PET/CT scan, prior to the initiation of\nphage therapy on September 23. The next scan was not performed until October 24, which was 18 days after the end of\nphage treatment. This delay makes it impossible to attribute radiologic improvement specifically to phages, as the observed\neffect may also reflect ongoing antibiotic therapy, immune responses, or natural recovery.\nClinically, the infection was not cured but managed through suppression, as shown by the patient’s continued need for oral\nfluoroquinolone therapy. Although the case demonstrates that phage therapy was safe and feasibly implemented, the lack of\npost-treatment microbiological biomarkers or mechanistic data prevents any firm conclusion that phages contributed to\nclinical improvement. This evidentiary gap is common across most compassionate use phage therapy reports.\nThe in vitro data showed reduced MICs and enhanced bacterial killing in the presence of phages, but the study did not use\nstandard synergy testing methods. It lacked checkerboard assays and/or time kill studies with set thresholds compared to the\nmost active single agent. In addition, the phage antibiotic combinations were not tested against appropriate controls. These\nomissions limit the strength of the claim that phage and antibiotic synergy drive clinical improvement.\nThe increase in inflammatory markers following phage therapy is described as mild and expected. However, from a critical\nstandpoint, the elevations in CRP and ESR, though transient, may represent a meaningful systemic immune response to\nintravenous phage administration, even in a clinically stable patient. This possibility was not explored in detail and deserves\nfurther investigation, especially in relation to immunogenicity and repeat dosing.\nThe assertion that phage therapy restored fluoroquinolone susceptibility is also not well supported. While post-treatment\nMICs were lower and the patient tolerated oral levofloxacin, the study did not include sequencing or resistance profiling of\npost-treatment isolates. Therefore, it is unclear whether the susceptible strain was a direct descendant of the fluoroquinolone\nresistant strains identified earlier. The change in susceptibility could reflect strain replacement, selection of a preexisting\nsusceptible subpopulation, or natural variation rather than true reversion of resistance.\nOverall, the innovation in this study lies in the operational and clinical framework for delivering phage therapy within a\nmodern infectious disease care model. That contribution is valuable and relevant for translation into real world practice.\nHowever, the mechanistic and pharmacodynamic claims regarding phage efficacy should be interpreted cautiously, given\nthe limitations in microbiological evidence, timing, and experimental rigor.\nMinor comments.\nWhile the study uses standard translational phage microbiology tools (spot testing, time-kill assays, biofilm assays), it is\nunclear whether these assays led to a meaningfully more effective treatment strategy than other possible phage cocktail\noptions in vivo. The authors should clarify how these in vitro results translated into the final clinical decision.\nLine 9: Proofreading is needed.\nLine 47: Replace “well-planned and well-timed” with “systematically planned and timely” to avoid redundancy.\nLines 60–61: The sentence describing post-phage therapy improvement is redundant (“reduced tracer uptake... no\nrecurrence of bacteremia” is repeated). Recommend condensing.\nLine 89: Change “bacterial eradication rate was achieved...” to “bacterial eradication was achieved in 6 of 7 cases...” for\nclarity.\nLines 95–98: The description of the patient’s complex surgical history could benefit from minor simplification or expanded\ndetail to improve readability and clinical context.\nLines 283–284: The phrase “slight increase in inflammatory markers” downplays the quantitative data (CRP 16.8 mg/L, ESR\n44 mm/h). Consider rephrasing as a “measurable increase” to reflect biological significance.\nLine 303: The report of “100% neutralization with sharp recovery” for PW21 is biologically implausible. Clarify whether this\nresult is due to technical variability, assay noise, or another artifact.\nLines 314–319: The sentence describing the phage therapy framework is overly long and contains multiple clauses.\nRecommend splitting into two sentences.\nLine 342: Rephrase “this is the index phage therapy case in Singapore” as “this represents the first documented case of\nphage therapy in Singapore”, especially in light of the surrounding discussion and critique of causality.\nLines 367–375: Suggest including a brief reference to standard definitions of synergy.\nLines 400–403: The discussion of cost savings is compelling. Consider adding a concluding sentence framing this as a\npotential health system benefit, especially in resource-limited or outpatient settings.\nFigure 2 – PET/CT Imaging. Add scale bars for spatial reference. Use consistent phrasing in captions to label timepoints\nclearly (e.g., “Pre-phage” [July], “Post-phage” [October]) to guide readers through the treatment timeline.\nFigure 3 –Define “LOD” (Limit of Detection).\nFigure 4 –Add caption labels to key steps in the workflow (e.g., TDM, iACT, Phage Screening). Visually distinguish parallel\nvs. sequential processes to clarify the integrated treatment approach.", "reviewer_2": "This is a nice exposition of a clinical case with a successful outcome. Initial failures are described by the authors as\npotentially attributable to inadequate antibiotic therapy. It seems that success followed further surgical debridement and\nimproved antibiotic management, as well as a course of bacteriophage therapy.\nThey also describe four members of what we presume to be a diversified population of P aeruginosa, including a shift in\nsusceptibility toward quinolones.\nSome of the assays are slightly unconventional (mixtures of bacteria, high phage MOIs etc) and the authors show that the\nphages were not highly active, even in combination, under the in vitro conditions tested. They show antibiotic synergy, which\nis interesting as it includes a quinolone and one (but not most) beta-lactams.\nIt would be ideal to show that the phages themselves contributed to outcome, or at least that some sort of amplification of\nphage numbers were detected as a surrogate for successful predation. The samples required to show this are not always\navailable, of course.\nIn order to enhance the manuscript, I have a number of more specific suggestions:\n1. The differences between the P aeruginosa isolates are not very clear to me at least, as only % identity, an ST, an\nantibiogram and a gene list are provided but these are presumably four members of a diversified population of bacteria\nunder stress. The CAZ-AVI and CIP synergy are interesting, as neither ab alone provides sustained growth suppression, nor\ndo any of the phage combinations. This would be worth a little more explanation, as it bears significantly on differential\nphage responsiveness. Relevant aspects to discuss are bacterial defence systems and prophages in particular I think.\n2. It would also be interesting to discuss the LEVO susceptibility shift in kinetics and contrast the mechanism of the CAZ AVI\neffect, since it has such a different mode of action. Do the bacterial genomic data explain this in any way?\n3. I think the supp data on phage-antibiotic synergy might be more strongly featured and discussed, including justification for\nthe slightly unusual choice to combine all 4 bacteria as a mixture in testing, and to use the very high MOI of 100. Are there\nany data at subMIC in support of synergy?\n4. Fig S9 shows no real suppression of growth by any phage combination beyond 6-8h, apparently using a very high MOI of\n100. Are there any data at lower MOIs?\n5. Sharp initial collapses (2-3 log) in control growth kinetics are not discussed in Figs S8-14. Could the authors please\ncomment on this and its impact on the readers interpretation of the time-kill curves.\n6. I saw no data regarding the presence of phages in blood – do we know what clearance kinetics were like?\n7. Do we have any evidence of phage amplification to show that phages were successfully preying on the bacterial\npopulation/s?\n8. Was there any evidence of a host inflammatory reaction to the phage administration?\n9. Were any (targeted) bacteria or (administered) phages recovered from wounds/ mediastinum/ tissue or body fluids from\nwhich we might learn about any co-adaptation? What was done to retrieve them (eg it may be that there were no\npseudomonas and no phages present soon after initial phage administration) ?\n10. The serum neutralization data are a little confusing as well – it seems that the serum was diluted to low levels (1%) and\nthe bacterial density was high (in blood, a 10e9 dose will be diluted immediately to 10e4-5 etc) – it might be better to set\nthese data aside completely or to add in more data/ revise that section significantly to make it more impactful/ informative.\n11. With regard to layout/ content:\na. Fig 4 is unnecessary I think, or could move to / combine with Fig S2\nb. Fig 3 could be simplified by omitting EMs and spot tests, and referring instead to Fig S3\nc. Overall, the text could be greatly reduced and the focus shifted to those questions that are raised – eg is it the case that\nphages which are apparently poor performers in vitro can be highly effective in vivo? And to highlight the evidence for this eg\nphage amplification, host responses, bacterial lysis products in blood etc.", "reviewer_3": "Dear Author,\nIn this Case Report to the Journal, you report on a young female patient with an early refractory thoracic vascular graft\ninfection following aortic root replacement and pulmonary homograft replacement that was successfully treated with a\ntargeted combination of phages and antimicrobial treatment. This report highlights the novel use of phage therapy in a case\nwhere a re-operation with complete removal of the infected prosthesis was deemed not possible due to a history of several\nre-sternotomies (5 in total) and a molesting biofilm-producing bacteria (Pseudomonas aeruginosa). After achieving\nmultidisciplinary consensus and following several bacteremia episodes under antimicrobial treatment, the authors opted to\nrapidly screen the patient for potential phage therapy and therapeutic drug monitoring. She was successfully treated with\nphages for two weeks, achieving inflammation control without the need for further intravenous antimicrobial treatment. At\nfollow-up, the patient remains on suppressive oral treatment.\nThe document is well written and the analysis as well as the conclusion are sound.\nFrom a clinical point of view, there are several questions to be answered, before accepting this interesting manuscript for\npublication.\nLine 106: Why was mediastinal lavage performed 6 days after diagnosis of mediastinitis, why not earlier? And what did you\nperform exactly?\n• Was a tissue flap (muscle/omental) coverage of the prosthesis considered? The group of Hannover showed that this\napproach was feasible when treating early thoracic vascular graft infections (Umminger et al, Eur Jour Cardiothorac Surg,\n2016)\n• What did you find intraoperatively, pus? Were intraoperative samples sent for culture? And if so, did they grow positive for\nthe same microorganism?\nLine 185: I agree with you that this patient was not operable due to the number of re-sternotomies and the acuity of the\ninfection and the last surgery. However, did you consider at any point to do a re-sternotomy with mediastinal-irrigation and\nflap-coverage instead of re-operation on cardiopulmonary bypass and explantation of the infected prosthesis?\nLine 195: “Definitive surgery to remove the composite vascular graft was not possible”. Again, I agree with you that the\npatient is not operable, but the wording might not be precise enough, since it is always possible.\nWhen and how was the last follow-up performed? Did you perform an FDG-PET/ CT to control tracer uptake and steer\nantimicrobial treatment?\nBest regards,"}, "author_response": "“Timely bespoke bacteriophage–antibiotic combination successfully treats refractory Pseudomonas aeruginosa\nmediastinitis and vascular graft infection.”\nPoint-by-Point Response to Reviewers Comments\nReviewer #1\nWe thank the reviewer for their positive assessment of the manuscript, particularly\nnoting the strengths of the multidisciplinary approach, early integration of phage\ntherapy, rigorous bacterial isolate screening, and therapeutic drug monitoring. We also\nappreciate the recognition that the innovation in this study lies in the operational and\nclinical framework for delivering phage therapy within a modern infectious disease\ncare model, which we agree is valuable and relevant for translation into real-world\npractice. We acknowledge the concerns regarding the circumstantial nature of the\nevidence for phage efficacy. Below, we provide a detailed, point-by-point response to\neach specific comment raised, including clarifications, additional analyses, and\nmanuscript revisions to address these limitations.\n[Point #1]\nThe patient was already showing improvement on the September 10 PET/CT scan,\nprior to the initiation of phage therapy on September 23. The next scan was not\nperformed until October 24, which was 18 days after the end of phage treatment. This\ndelay makes it impossible to attribute radiologic improvement specifically to phages,\nas the observed effect may also reflect ongoing antibiotic therapy, immune responses,\nor natural recovery.\nWe thank the reviewer for this important observation regarding the timing of imaging\nand its impact on causal inference. We agree that the evidence is circumstantial and\nthat radiologic improvement cannot be attributed to phage therapy with absolute\ncertainty. However, PET/CT findings provide additional context that supports a likely\nbeneficial contribution of phage therapy.\nSpecifically, despite prolonged high-dose antibiotic therapy since April 2024, tracer\nuptake around the aortic graft persisted. Between May 2024 (Fig. 2C) and July 2024\n(Fig. 2D), the modest reduction in uptake was temporally associated with antibiotic\ndose escalation in June 2024. From July 2024 to September 2024 (Fig. 2E), tracer\nuptake around the graft increased despite continued high-dose piperacillin–\ntazobactam and levofloxacin, suggesting limited efficacy of antibiotics alone against\nthis likely biofilm-driven process. Reduction in tracer uptake around the graft was only\nobserved following initiation of phage therapy in September 2024, as documented in\nthe October 2024 PET/CT scan.\nWe have revised Figure 2 to include an updated April 2025 PET/CT demonstrating\nsustained improvement on levofloxacin monotherapy (despite only intermediate\nsusceptibility of P. aeruginosa to fluoroquinolones). The nuclear medicine\ninterpretation, corroborated by our co-author and clinical specialist who reviewed the\nserial scans, supports a likely contributory role of phage therapy in the observed\nradiological improvement. The figure legend has been updated to reflect this\ninterpretation and temporal sequence.\n“Timely bespoke bacteriophage–antibiotic combination successfully treats refractory Pseudomonas aeruginosa\nmediastinitis and vascular graft infection.”\nAlthough we cannot definitively attribute the patient’s improvement to phage therapy\nalone, its use was a deliberate, multidisciplinary decision aimed at addressing the\nongoing biofilm-associated infection that had not resolved with antibiotics. The\npatient's subsequent clinical stabilisation and radiologic improvement, in conjunction\nwith in vitro synergy data and therapeutic monitoring, suggest a likely contributory role\nof phage therapy to the observed outcome.\nModifications to the manuscript:\n- Figure 2, page 9\n- Legend of Figure 2, page 10\n[Point #2]\nClinically, the infection was not cured but managed through suppression, as shown by\nthe patient’s continued need for oral fluoroquinolone therapy. Although the case\ndemonstrates that phage therapy was safe and feasibly implemented, the lack of post-\ntreatment microbiological biomarkers or mechanistic data prevents any firm\nconclusion that phages contributed to clinical improvement. This evidentiary gap is\ncommon across most compassionate use phage therapy reports.\nWe thank the reviewer for this comment and appreciate the opportunity to provide\nadditional clinical context. Despite receiving appropriate culture-directed antibiotic\ntherapy in April–May 2024, the patient experienced breakthrough P. aeruginosa\nbacteremias, documented on two occasions through frequent surveillance blood\ncultures (without waiting for clinical symptoms manifestation) performed due to the\ncomplexity of her case. Clinical symptoms were minimal, and inflammatory markers\nremained within normal limits even during these episodes of confirmed bacteremia,\nunderscoring the muted biochemical and clinical responses that complicated\nassessment of treatment efficacy (Fig. 1).\nSerial PET/CT imaging, however, demonstrated increasing tracer uptake around the\naortic valve prosthesis despite high-dose antibiotics, consistent with persistent deep-\nseated infection. Following initiation of phage therapy, we observed (1) radiological\nimprovement on PET/CT, and (2) no recurrence of bacteremia on surveillance cultures\nthrough April 2025, despite the patient being maintained on levofloxacin monotherapy\nsince December 2024 (with P. aeruginosa isolates of intermediate fluoroquinolone\nsusceptibility). We have updated Figure 2 to reflect this longitudinal imaging.\nIn cases such as this—vascular graft infections with recurrent bacteremia not\namenable to surgical replacement—long-term suppressive antibiotic therapy is\nstandard practice and does not necessarily represent failure of phage therapy. The\ndecision to continue suppression is made on an individual basis through shared\ndecision-making with the patient, given the high risk associated with treatment\nwithdrawal.\n“Timely bespoke bacteriophage–antibiotic combination successfully treats refractory Pseudomonas aeruginosa\nmediastinitis and vascular graft infection.”\nFinally, while we lack post-treatment microbiologic biomarkers, we note and agree that\nthis constraint is typical of compassionate-use protocols, where invasive sampling is\nclinically contra-indicated. Similarly, while mechanistic confirmation of phage activity\nin vivo is commonly difficult to demonstrate, we provided additional in vitro data that\nestablish a mechanistic basis for the synergistic effect of the phage and antibiotic\ncombination (see next point).\nModifications to the manuscript:\n- We have revised Figure 1, and the Legend of Figure 1 on pages 7 and 8,\nrespectively for clarity.\n[Point #3]\nThe in vitro data showed reduced MICs and enhanced bacterial killing in the presence\nof phages, but the study did not use standard synergy testing methods. It lacked\ncheckerboard assays and/or time kill studies with set thresholds compared to the\nmost active single agent. In addition, the phage antibiotic combinations were not tested\nagainst appropriate controls. These omissions limit the strength of the claim that phage\nand antibiotic synergy drive clinical improvement.\nWe thank the reviewer for this important observation. We agree that the initial synergy\ntesting lacked the full range of standardized methodologies. In response to this\nfeedback, we have now conducted additional experiments incorporating time-kill\ncurves using phage alone, antibiotic alone, and their combinations, with clearly defined\nsynergy thresholds (≥2 log₁ ₀ increase in killing at sub-MIC concentration). These new\ndata have been included in the revised manuscript (Figure 4) and demonstrate\nreproducible synergy between the phage cocktails and key antibiotics against the\npatient isolates. Further to this, we tested our in-house P. aeruginosa transposon\nmutants Tn::mexB, deficient for the MexAB-OprM membrane transporter complex,\nand showed that it constitutes a key determinant of efficacy for several of our phages\ni.e. the receptor for these phages. MexAB-OprM is an efflux transporter that has been\nshown to confer resistance to penicillins and quinolones. We (and others - see\nreferences below) have shown that the disruption of MexAB-OprM leads to\nsensitization to these antibiotics We believe these additional findings strengthens the\nmechanistic foundation for the observed clinical outcome and enhances the\ntranslational value of our findings. We have incorporated these findings in Figure 3\nand Supplementary Figures.\nReferences:\n- Chan, B., Sistrom, M., Wertz, J. et al. Phage selection restores antibiotic\nsensitivity in MDR Pseudomonas aeruginosa. Sci Rep 6, 26717 (2016).\nhttps://doi.org/10.1038/srep26717\n- Ho P, Dam LC, Koh WRR, Nai RS, Nah QH, Rajaie Fizla FBM, Chan CC, Aung\nTT, Goh SG, Fang Y, Lim Z, Koh MG, Demott M, Boucher YF, Malleret B, Gin\nKY, Dedon P, Moreira W. Screening of the PA14NR Transposon Mutant Library\n“Timely bespoke bacteriophage–antibiotic combination successfully treats refractory Pseudomonas aeruginosa\nmediastinitis and vascular graft infection.”\nIdentifies Genes Involved in Resistance to Bacteriophage Infection in\nPseudomomas aeruginosa. Int J Mol Sci. 2024 Jun 26;25(13):7009. doi:\n10.3390/ijms25137009.\nModifications to the manuscript:\n- We created a Figure 3 (Phage Therapy Workflow) on page 13, and revised Figure\n4 (which was previously figure 3) and its legend on page 16 and 17, respectively\n- Figure 4 describes the mechanisms behind antibiotic-sensitization, MIC shift and\nincreased killing at sub-MIC of antibiotics.\n- Additional Supplementary Figure S17 showing the role of MexAB-OprM in phage\nefficacy\n- Results section, pages 14-17, lines now 363-462.\n- Discussion section, page 19-20, lines 531-558.\n[Point #4]\nThe increase in inflammatory markers following phage therapy is described as mild\nand expected. However, from a critical standpoint, the elevations in CRP and ESR,\nthough transient, may represent a meaningful systemic immune response to\nintravenous phage administration, even in a clinically stable patient. This possibility\nwas not explored in detail and deserves further investigation, especially in relation to\nimmunogenicity and repeat dosing.\nWe agree that the transient rise in CRP and ESR could reflect a systemic immune\nresponse to intravenous phage therapy. However, the patient remained clinically\nstable and asymptomatic throughout the course of Phage Therapy, and no clinical\nevent correlated with the CRP and ESR trends. Nonetheless, we have now expanded\nthe discussion to address potential immunogenicity and the need for additional study\nin future cases, particularly in the context of repeat dosing and immunological markers.\nModification to manuscript:\n- Discussion section, page 20, lines 560-562.\n[Point #5]\nThe assertion that phage therapy restored fluoroquinolone susceptibility is also not\nwell supported. While post-treatment MICs were lower and the patient tolerated oral\nlevofloxacin, the study did not include sequencing or resistance profiling of\npost-treatment isolates. Therefore, it is unclear whether the susceptible strain was a direct\ndescendant of the fluoroquinolone resistant strains identified earlier. The change in\nsusceptibility could reflect strain replacement, selection of a preexisting susceptible\nsubpopulation, or natural variation rather than true reversion of resistance.\nWe thank the reviewer for highlighting this important point. Considering this and as\ndiscussed above, we used a P. aeruginosa MexAB-OprM receptor-deficient mutant\nand tested its susceptibility to our phage cocktail. The mexAB-OprM complex has been\n“Timely bespoke bacteriophage–antibiotic combination successfully treats refractory Pseudomonas aeruginosa\nmediastinitis and vascular graft infection.”\npreviously been identified (see Point #3) as a major receptor for P. aeruginosa phages.\nIts deletion leads to increased sensitivity to Fluoroquinolones and Beta-lactams.\nPhages targeting the MexAB-OprM complex have been used both in vitro and in vivo\nand achieved phenotypic trade-off between antibiotic resistance and phage sensitivity.\nWe tested the ability of the phages used in our therapeutic cocktail to infect this mutant.\nThe results showed that 1 out of 3 of the phages composing our therapeutic cocktail\nrely on MexAB-OprM to carry out a lytic infection. These findings provide a mechanistic\nrational for the antibiotic sensitization observed in vitro, and likely in vivo. We have\nrevised the manuscript to present these critical findings (Figure 4 and supplementary\nFigure S17). We also acknowledge the need of post-treatment isolate characterization\nexplicitly and propose genomic follow-up as a critical component for future studies.\nModification to the manuscript:\n- Figure 4\n- Additional Supplementary Figure S17 showing the role of MexAB-OprM in phage\nefficacy\n- Discussion section, page 20, lines 553-555.\n[Point #6]\nWhile the study uses standard translational phage microbiology tools (spot testing,\ntime-kill assays, biofilm assays), it is unclear whether these assays led to a\nmeaningfully more effective treatment strategy than other possible phage cocktail\noptions in vivo. The authors should clarify how these in vitro results translated into the\nfinal clinical decision.\nWe thank the reviewer for this valuable observation. The in vitro assays (particularly\nthe phage/antibiotic combination kill kinetic and biofilm disruption assays) were used\nto prioritize phages with complementary activity profiles, including rapid lytic kinetics,\nefficacy against biofilm-embedded cells, and performance in combination with the\npatient's antibiotics. These results informed the clinical decision to select a cocktail\ntargeting both planktonic and biofilm-associated bacterial populations. However, we\nacknowledge that in the context of a single compassionate-use case, it is not possible\nto definitively determine whether this combination was the most effective among all\npossible phage and antibiotics combinatorial options. We have clarified this\nuncertainty in the revised manuscript (revised Figure 3 describing our phage therapy\nworkflow) and explicitly linked the in vitro findings to the clinical selection process\n(Figure 4 linking the phage selection process to the clinical treatment decision)\nModification to the manuscript:\n- Revised Figure 3 and Legend of Figure 3; Results section, page 13\n- Revised Figure 4 and Legend of Figure 4; Results section, page 16 -17 ;\nDiscussion section, page 19 lines 528-530\n“Timely bespoke bacteriophage–antibiotic combination successfully treats refractory Pseudomonas aeruginosa\nmediastinitis and vascular graft infection.”\nMinor comments.\nLine 9: Proofreading is needed. Thank you. We revised it.\nLine 47: Replace “well-planned and well-timed” with “systematically planned and\ntimely” to avoid redundancy. Thank you for the recommendation. The text has been\nrevised. Please see line now 49.\nLines 60–61: The sentence describing post-phage therapy improvement is redundant\n(“reduced tracer uptake... no recurrence of bacteremia” is repeated). Recommend\ncondensing.\nWe have revised the sentences to the following: “Post phage therapy, radiological\nimprovements were seen on serial PET/CT and bacteremia was eliminated, without\nrecurrence. The patient was transitioned to an oral fluoroquinolone maintenance\ntreatment” in lines 56-58\nLine 89: Change “bacterial eradication rate was achieved...” to “bacterial eradication\nwas achieved in 6 of 7 cases...” for clarity. The statement has been revised as\nrecommended in lines now 102-103.\nLines 95–98: The description of the patient’s complex surgical history could benefit\nfrom minor simplification or expanded detail to improve readability and clinical context.\nTo improve clarity, we have revised the patient’s complex surgery as follow in lines\nnow 106-113: “A 36-year-old female patient with a history of congenital heart disease\n(pulmonary atresia with ventricular septal defect) with 4 prior sternotomies for\ncorrective heart surgery developed progressive dilatation of her ascending aorta with\nsevere aortic regurgitation and pulmonary homograft stenosis in 2023. This was\nassociated with admissions for decompensated heart failure. To address this, she\neventually underwent a Bentall procedure where the affected aortic valve, aortic root\nand ascending aorta were replaced by a composite vascular graft and the pulmonary\nhomograft was also replaced in the same setting on January 25th, 2024, just 5.5 weeks\nafter she had delivered her second child.”\nLines 283–284: The phrase “slight increase in inflammatory markers” downplays the\nquantitative data (CRP 16.8 mg/L, ESR 44 mm/h). Consider rephrasing as a\n“measurable increase” to reflect biological significance. The statement has been\nrephrased in line 312.\nLine 303: The report of “100% neutralization with sharp recovery” for PW21 is\nbiologically implausible. Clarify whether this result is due to technical variability, assay\nnoise, or another artifact.\nWe thank this reviewer for this observation. We believe that these results are due to\ntechnical variability or inadequacy and, in accordance with reviewer 2 who has raised\na similar point and suggested to leave these results out, we propose to remove these\nfindings in accordance with reviewer 2’s suggestion.\nModifications to the manuscript:\n- Removal of Supplementary Figure S16 and its description.\n- Removal of the immune neutralisation description of phages before and during\nphage therapy in previous page 20 of the first submitted manuscript version.\n- Discussion section Page 20 now : We have added a comment on the need for\nimmunological studies on phage neutralisation throughout PT in lines 567.\n“Timely bespoke bacteriophage–antibiotic combination successfully treats refractory Pseudomonas aeruginosa\nmediastinitis and vascular graft infection.”\nLines 314–319: The sentence describing the phage therapy framework is overly long\nand contains multiple clauses. Recommend splitting into two sentences.\nWe have revised the sentences in lines now 510-530. The revised section is as follows:\n“Despite months of high-dose, TDM-guided antibiotic therapy with iACT optimisation,\nserial 18F-FDG PET/CT scans demonstrated persistent tracer uptake on the aortic\ngraft, suggesting likely long-term treatment failure (…). Adjunctive phage therapy was\nconsidered early (i.e. before deterioration) in April 2024 (...). In this report, we propose\na framework for the timely systematic incorporation of compassionate adjunctive\nphage therapy (…).”\nLine 342: Rephrase “this is the index phage therapy case in Singapore” as “this\nrepresents the first documented case of phage therapy in Singapore”, especially in\nlight of the surrounding discussion and critique of causality.\nWe have revised line 342 (now 528-529 ) to “…..marking Singapore’s first reported\nclinical phage use”.\nLines 367–375: Suggest including a brief reference to standard definitions of synergy.\nBasic definition (synergy = >2-log increased killing) with the references below are\nadded to Method section page 24 lines 664-671.\nReferences:\n- Comeau AM, Tétart F, Trojet SN, Prère M-F, Krisch HM (2007) Phage-Antibiotic\nSynergy (PAS): β-Lactam and Quinolone Antibiotics Stimulate Virulent Phage Growth.\nPLoS ONE 2(8): e799. https://doi.org/10.1371/journal.pone.0000799\n- Morris TC, Reyneke B, Khan S, Khan W. Phage-antibiotic synergy to combat\nmultidrug resistant strains of Gram-negative ESKAPE pathogens. Sci Rep. 2025 May\n18;15(1):17235. doi: 10.1038/s41598-025-01489-y. PMID: 40383795; PMCID:\nPMC12086229.\n- Nicolle P, Faguet M. La synergie lytique de la pénicilline et du bactériophage, étudiée\nau microbiophotomètre [The lytic synergy of penicillin and bacteriophage, studied with\na microbiophotometer]. Ann Inst Pasteur (Paris). 1947 May;73(5):490-5. French.\nPMID: 20253582.\nLines 400–403: The discussion of cost savings is compelling. Consider adding a\nconcluding sentence framing this as a potential health system benefit, especially in\nresource-limited or outpatient settings.\nWe thank the reviewer for this positive comment. In the section conclusion, this is\ncommented and concluded in lines now 601-604\nFigure 2 – PET/CT Imaging. Add scale bars for spatial reference. Use consistent\nphrasing in captions to label timepoints clearly (e.g., “Pre-phage” [July], “Post-phage”\n[October]) to guide readers through the treatment timeline.\nFigure 2 has been amended to include representative PET-CT images in Apr 2025.\nThe timepoint labels are also revised based on the above recommendations. A scale\nbar has also been added.\nFigure 3 –Define “LOD” (Limit of Detection).\n“Timely bespoke bacteriophage–antibiotic combination successfully treats refractory Pseudomonas aeruginosa\nmediastinitis and vascular graft infection.”\nThe limit of detection (LOD) in a time kill assay here refers to the lowest bacterial cell\nconcentration that can be reliably detected using plate count method. Definition has\nbeen added.\nFigure 4 –Add caption labels to key steps in the workflow (e.g., TDM, iACT, Phage\nScreening). Visually distinguish parallel (color coded) vs. sequential processes\n(directed by arrows) to clarify the integrated treatment approach.\nWe have significantly revised Figure 4 (now Figure 5) to reflect the reviewer`s\nsuggestions. Figure 5 now presents 3 tracks (Patient Journey, Clinical Process and\nPhage Pipeline) which illustrate the integrated treatment approach, with detailed\nsequential and parallel processes and arrows linking each tracks.\nModification to the manuscript:\n- New Figure 5 (ex-Figure 4), page 22\nReviewer #2\n[Point #1]\nThis is a nice exposition of a clinical case with a successful outcome. Initial failures\nare described by the authors as potentially attributable to inadequate antibiotic\ntherapy. It seems that success followed further surgical debridement and improved\nantibiotic management, as well as a course of bacteriophage therapy. They also\ndescribe four members of what we presume to be a diversified population of P\naeruginosa, including a shift in susceptibility toward quinolones.Some of the assays\nare slightly unconventional (mixtures of bacteria, high phage MOIs etc) and the\nauthors show that the phages were not highly active, even in combination, under the\nin vitro conditions tested. They show antibiotic synergy, which is interesting as it\nincludes a quinolone and one (but not most) beta-lactams. It would be ideal to show\nthat the phages themselves contributed to outcome, or at least that some sort of\namplification of phage numbers were detected as a surrogate for successful predation.\nThe samples required to show this are not always available, of course.\nWe thank the reviewer for their positive evaluation of the case presentation and for\nrecognizing the interesting aspects of the P. aeruginosa population structure and\nobserved antibiotic synergy. We appreciate the constructive suggestions to improve\nthe manuscript, particularly regarding clarification of isolate differences, further\ndiscussion of susceptibility shifts, and consideration of additional data on phage\nactivity and host response. Below, we provide a detailed, point-by-point response to\neach of these comments, including clarifications, additional discussion, supplementary\nexperiments and additional data.\nIn order to enhance the manuscript, I have a number of more specific suggestions:\n[Point #2]\nThe differences between the P aeruginosa isolates are not very clear to me at least,\nas only % identity, an ST, an antibiogram and a gene list are provided but these are\npresumably four members of a diversified population of bacteria under stress. The\n“Timely bespoke bacteriophage–antibiotic combination successfully treats refractory Pseudomonas aeruginosa\nmediastinitis and vascular graft infection.”\nCAZ-AVI and CIP synergy are interesting, as neither ab alone provides sustained\ngrowth suppression, nor do any of the phage combinations. This would be worth a little\nmore explanation, as it bears significantly on differential phage responsiveness.\nRelevant aspects to discuss are bacterial defence systems and prophages in particular\nI think.\nGood point, we checked both and added; no link with synergy BUT synergy\nmechanistic explanation detailed in next point below.\nModifications to the manuscript:\n- Suppl. Figure S1 showing Phage Defense Systems identified in patient isolates’\ngenomes.\n- Supplementary Table S4, added prophages characterisation showing that they are\ndevoid of VF, AMR genes or other remarkable genetic elements.\n- Results section, page 12, lines 277-279. “The prophages and Phage Defense\nSystems (PDS) in these 4 isolates were identified and characterised (described in\nsupplementary information). Prophages are devoid of VF, AMR genes or other\nremarkable genetic elements.”\n[Point #3]\nIt would also be interesting to discuss the LEVO susceptibility shift in kinetics and\ncontrast the mechanism of the CAZ AVI effect, since it has such a different mode of\naction. Do the bacterial genomic data explain this in any way?\nWe thank the reviewer for raising this important point. LEVO, PTZ or CZA (we note\nthat the reviewer mentioned CIP. However, we have only used LEVO as the only\nfluoroquinolone) have distinct mechanisms of action, and their effects in combination\nwith phage therapy merit deeper mechanistic discussion. We provide additional\nevidence of a mechanistic basis for the phage-driven antibiotic sensitisation. We used\na P. aeruginosa MexAB-OprM receptor-deficient mutant and tested its susceptibility to\nour phage cocktail. The mexAB-OprM complex has previously been identified (by us\nand others, see references below) as a major receptor for P. aeruginosa phages. Its\ndeletion leads to increased sensitivity to Fluoroquinolones, Beta-lactams and\nCephalosporins. Phages targeting the MexAB-OprM complex have been used both in\nvitro and in vivo and achieved a phenotypic trade-off between antibiotic resistance and\nphage sensitivity. We tested the ability of the phages used in our therapeutic cocktail\nto infect this mutant. The results showed that 1 out of 3 of the phages composing our\ntherapeutic cocktail rely on MexAB-OprM to carry out a lytic infection. These findings\nprovide a mechanistic rationale for the antibiotic sensitisation observed in vitro and\nlikely in vivo. We have revised the manuscript to present these critical findings (Figure\n3 and supplementary Figure SXX ).\nReferences:\n“Timely bespoke bacteriophage–antibiotic combination successfully treats refractory Pseudomonas aeruginosa\nmediastinitis and vascular graft infection.”\n- Chan, B., Sistrom, M., Wertz, J. et al. Phage selection restores antibiotic\nsensitivity in MDR Pseudomonas aeruginosa. Sci Rep 6, 26717 (2016).\nhttps://doi.org/10.1038/srep26717\n- Ho P, Dam LC, Koh WRR, Nai RS, Nah QH, Rajaie Fizla FBM, Chan CC, Aung\nTT, Goh SG, Fang Y, Lim Z, Koh MG, Demott M, Boucher YF, Malleret B, Gin\nKY, Dedon P, Moreira W. Screening of the PA14NR Transposon Mutant Library\nIdentifies Genes Involved in Resistance to Bacteriophage Infection in\nPseudomomas aeruginosa. Int J Mol Sci. 2024 Jun 26;25(13):7009. doi:\n10.3390/ijms25137009\n- Modifications to the manuscript:\n- Revised Figure 4, Legend of Figure 4\n- Additional Supplementary Figure S17 showing the role of MexAB-OprM in phage\nefficacy\n[Point #4]\nI think the supp data on phage-antibiotic synergy might be more strongly featured\nand discussed, including justification for the slightly unusual choice to combine all 4\nbacteria as a mixture in testing, and to use the very high MOI of 100. Are there any\ndata at subMIC in support of synergy?\nWe thank the reviewer for this insightful comment. The decision to combine all four\nisolates and use a high MOI of 100 was driven by the clinical context of a polymicrobial,\nbiofilm-associated infection involving genetically related but phenotypically variable\nstrains. This approach aimed to simulate in vivo complexity and ensure maximal killing\nacross the population. However, we acknowledge that using a mixture of 4 bacteria is\nan unusual experimental design and have now revised the manuscript to explicitly\njustify this approach. Additionally, we have conducted additional experiments using\nlower MOIs, and sub-MIC antibiotic concentrations, and included these data in the\nrevised manuscript. Our data shows a clear MIC shift for LEVO and PTZ leading to\nincreased killing (>2-log) at sub-MIC concentration. We agree with the reviewer that\nour data showing phage-antibiotic synergy should be more strongly featured and\ndiscussed. We have now incorporated them in revised Figure 3 and discuss them\naccordingly.\nModifications to the manuscript:\n- Results section, pages 15-16. Revised Figure 4.\n- Included the justification of combining 4 bacteria isolates in Legend of Figure 4\nin page 16 of Results section.\n[Point #5]\nFig S9 shows no real suppression of growth by any phage combination beyond 6-8h,\napparently using a very high MOI of 100. Are there any data at lower MOIs?\n“Timely bespoke bacteriophage–antibiotic combination successfully treats refractory Pseudomonas aeruginosa\nmediastinitis and vascular graft infection.”\nWe agree with the reviewer that single-phage, and the combined-phage treatment did\nnot lead to sustained killing over time. We provide additional evidence at a lower MOI\nof 1.\nModification to the manuscript:\n- Additional Supplementary Figure S15\n[Point #6]\nSharp initial collapses (2-3 log) in control growth kinetics are not discussed in Figs S8-\n14. Could the authors please comment on this and its impact on the readers\ninterpretation of the time-kill curves.\nWe thank the reviewer for this important observation. The sharp initial drops in\nbacterial density observed in the controls (Figs S8–S14) are likely attributable to sub-\noptimal growth conditions for that set of experiments. Importantly, these effects were\nobserved across both treated and untreated conditions and did not differentially impact\nthe interpretation of phage-antibiotic synergy. We have now included this clarification\nin the revised figure legends in the relevant parts of the supplementary information.\nWe have also repeated these kill kinetic experiments and showed that these initial\ncollapses were not reproduced and did not affect the interpretation of our findings.\nModifications to the manuscript:\n- New Figure 4A in Results section, page 16.\n- New Figure S11-S14\n- Supplementary Figure S8-S10 – The comment “The sharp initial drops in bacterial\ndensity observed in the control are likely attributable to sub-optimal growth\nconditions for that set of experiments” is included in every Legend of Figure S8-\nS10.\n- Additional Suppl figure S15 (low MOI)\n[Point #7]\nI saw no data regarding the presence of phages in blood – do we know what clearance\nkinetics were like?\nWe thank the reviewer for this observation. We have now included additional data\nshowing that we can detect phages DNA in the patient blood sample at day 8 and day\n15 of the phage therapy course. While we were able to detect phage DNA, we are not\nable to quantify phage clearance in that case. We have discussed this point in our\nrevised manuscript.\nModifications to the manuscript:\n- Supplementary information. Figure S18\n- Introduction section – Figure 1 (page 7) and Legend of Figure 1 (page 8 line 153 –\n155 )– The phrase “ (…) PT, Phage Therapy; Red blood drop symbols during PT,\n“Timely bespoke bacteriophage–antibiotic combination successfully treats refractory Pseudomonas aeruginosa\nmediastinitis and vascular graft infection.”\nindicate dates at which blood sample were taken to determine phage presence in\npatient blood (day 0, 8 and 15) (…).”\n- Results section Page 18, lines 489-490: The statement “Phage were detected in\npatient blood samples at day 8 and day 15 of phage therapy by PCR using primers\ndesigned to discriminate between the three phages (Supplementary Information).”\n- Discussion section, page 20, lines 567-569\n[Point #8]\nDo we have any evidence of phage amplification to show that phages were\nsuccessfully preying on the bacterial population/s?\nWe thank the reviewer for raising this important point. A PCR (polymerase chain\nreaction) test performed on patient blood samples at day 8 and day 15 of phage\ntherapy confirmed the presence of administered phages. While this supports phage\npersistence during treatment, we acknowledge that we do not have direct evidence of\nin vivo amplification. Demonstrating amplification was challenging given the limited\nnumber of post-therapy patient samples available. We have clarified this limitation in\nthe revised manuscript.\nModifications to the manuscript:\n- Same as previous point\n- Discussion section, we have inserted “PCR confirmed phage persistence in blood\nat days 8 and 15, though in vivo amplification could not be demonstrated due to\nlimited sampling.” in page 20, lines 567-569.\n[Point 9]\nWas there any evidence of a host inflammatory reaction to the phage administration?\nThe patient tolerated phage therapy well with no adverse events during treatment.\nBiochemically, we noted a measurable increase in the inflammatory markers; the\npreviously normal CRP and erythrocyte sedimentation rate (ESR) increased to a peak\nof 16.8 mg/L, and 44 mm/h, respectively, after phage administration, as described in\npage 18, lines 470-473\nClinically, the patient exhibited no signs or symptoms indicative of systemic\ninflammation or anaphylactic responses. However, slight increases in CRP and ESR,\nas previously noted, alluded to a mild inflammatory response post phage\nadministration, as described in page 18 lines 485-489\n[Point 10]\nWere any (targeted) bacteria or (administered) phages recovered from wounds/\nmediastinum/ tissue or body fluids from which we might learn about any co-\n“Timely bespoke bacteriophage–antibiotic combination successfully treats refractory Pseudomonas aeruginosa\nmediastinitis and vascular graft infection.”\nadaptation? What was done to retrieve them (eg it may be that there were no\npseudomonas and no phages present soon after initial phage administration) ?\nWe thank the reviewer for this question. No P. aeruginosa or administered phages\nwere recovered from mediastinal tissue or other body fluids after initiation of phage\ntherapy. The absence of recovery likely reflects both the improvement effect due to\nongoing targeted antimicrobial therapy and the limited sampling opportunities inherent\nto the clinical setting. However, PCR testing of patient blood at day 8 and day 15 of\nphage therapy confirmed the presence of administered phages, indicating persistence\nin vivo during treatment.\n[Point 11]\nThe serum neutralization data are a little confusing as well – it seems that the serum\nwas diluted to low levels (1%) and the bacterial density was high (in blood, a 10e9\ndose will be diluted immediately to 10e4-5 etc) – it might be better to set these data\naside completely or to add in more data/ revise that section significantly to make it\nmore impactful/ informative.\nWe agree with the reviewer that the serum neutralisation data, as presented, are\ninconclusive and confusing. Based on your recommendation and aligned with\nReviewer 1’s similar point, we have removed this section from the revised manuscript\nto avoid overinterpretation.\nWith regard to layout/ content:\n[Point 12]\na. Fig 4 is unnecessary I think, or could move to / combine with Fig S2\nWe have revised Figure 4 (now Figure 5) in accordance with Reviewer 1’s comments)\nto illustrate the parallel tracks of Patient Journey, Clinical Workflow and Phage\nMicrobiology workflow.\n[Point 13]\nb. Fig 3 could be simplified by omitting EMs and spot tests, and referring instead to\nFig S3. We agree and have revised Figure 3 significantly (now Figure 4)\n[Point 14]\nc. Overall, the text could be greatly reduced and the focus shifted to those questions\nthat are raised – eg is it the case that phages which are apparently poor performers in\nvitro can be highly effective in vivo? And to highlight the evidence for this eg\nphage amplification, host responses, bacterial lysis products in blood etc.\nWe thank the reviewer for this constructive feedback. We have significantly revised\nour main manuscript to highlight that phage + antibiotics are highly effective both in\nvitro and in vivo as either one of the modality alone is poorly effective. The critically\nimportant additional findings and modifications include:\n“Timely bespoke bacteriophage–antibiotic combination successfully treats refractory Pseudomonas aeruginosa\nmediastinitis and vascular graft infection.”\n- Results section pages 16-17, Figure 4 C-E: Antibiotic/phage synergy, MIC shift,\nSub-MIC increased killing and Biofilm activity\n- Supplementary Figure S17: Role of the MexAB-OprM efflux pump in phage\nsusceptibility\n- Supplementary Figure S18: Detection of Therapeutic Phages in Patient Blood\nReviewer #3\nIn this Case Report to the Journal, you report on a young female patient with an early\nrefractory thoracic vascular graft infection following aortic root replacement and\npulmonary homograft replacement that was successfully treated with a targeted\ncombination of phages and antimicrobial treatment.\nThis report highlights the novel use of phage therapy in a case where a re-operation\nwith complete removal of the infected prosthesis was deemed not possible due to a\nhistory of several re-sternotomies (5 in total) and a molesting biofilm-producing\nbacteria (Pseudomonas aeruginosa). After achieving multidisciplinary consensus and\nfollowing several bacteremia episodes under antimicrobial treatment, the authors\nopted to rapidly screen the patient for potential phage therapy and therapeutic drug\nmonitoring. She was successfully treated with phages for two weeks, achieving\ninflammation control without the need for further intravenous antimicrobial treatment.\nAt follow-up, the patient remains on suppressive oral treatment. The document is well\nwritten and the analysis as well as the conclusion are sound. From a clinical point of\nview, there are several questions to be answered, before accepting this interesting\nmanuscript for publication.\nWe thank the reviewer for their positive assessment of the manuscript, particularly\nnoting the novelty of phage therapy use in this complex, non-operable vascular graft\ninfection and the soundness of the analysis and conclusions. We appreciate the\nclinically focused questions raised and provide point-by-point responses below.\n[Point 1]\nLine 106: Why was mediastinal lavage performed 6 days after diagnosis of\nmediastinitis, why not earlier? And what did you perform exactly?\nFor clarity, we wish to emphasise that the onset of mediastinitis in this case was\ninsidious. The patient first developed fever on post-operative day (POD) 2, initially\nmanaged as culture-negative nosocomial pneumonia. On POD 6, a significant left\npleural effusion was identified and drained on POD 7. It was not until POD 10 that a\ncomplicated infection was suspected, when pleural drain output became cloudy. Due\nto persistent fever and P. aeruginosa bacteraemia commencing on POD 10, a CT\nthorax was performed on POD 13, confirming mediastinitis.\n“Timely bespoke bacteriophage–antibiotic combination successfully treats refractory Pseudomonas aeruginosa\nmediastinitis and vascular graft infection.”\nThe diagnostic work-up was necessarily sequential, with definitive treatment instituted\nas new findings emerged. Management was further complicated by a concurrent left\ngroin haematoma requiring evaluation, anaemia necessitating transfusion, the need\nfor patient and family consent for reoperation, and emergency operating theatre\nwaitlist constraints. The interval from diagnosis of mediastinitis to surgical intervention\nwas three days, not six as might be inferred. Figure 1 has been revised to clarify this\ntimeline; however, certain finer clinical details have been omitted from the manuscript\nto maintain focus.\nOperatively, the procedure involved reopening via the prior sternotomy, removal of\nsternal wires, and drainage of frank pus from the sternum, which on culture yielded P.\naeruginosa. The mediastinum was thoroughly irrigated using pulsavac, with\nadhesiolysis to prevent re-collection. Following irrigation, the mediastinum appeared\nsubstantially cleaner, and sternal rewiring with routine subcutaneous closure was\nperformed. For simplicity, we have described this as a mediastinal washout. The\noperative details were provided by the attending cardiothoracic surgeon, who is a co-\nauthor of this manuscript.\n[Point 2]\nWas a tissue flap (muscle/omental) coverage of the prosthesis considered? The group\nof Hannover showed that this approach was feasible when treating early thoracic\nvascular graft infections (Umminger et al, Eur Jour Cardiothorac Surg, 2016)\nThere was significant pus in the mediastinum and the patient was still bacteremic at\ntime of surgery. Tissue flap coverage was not considered by our cardiothoracic\nsurgeons.\n[Point 3]\nWhat did you find intraoperatively, pus? Were intraoperative samples sent for culture?\nAnd if so, did they grow positive for the same microorganism?\nYes, as described above. The intra-operative pus was positive for P. aeruginosa and\nthis was also described in the manuscript and presented in Figure 1 (we have revised\nthe figure for clarity).\n[Point 4]\nLine 185: I agree with you that this patient was not operable due to the number of re-\nsternotomies and the acuity of the infection and the last surgery. However, did you\nconsider at any point to do a re-sternotomy with mediastinal-irrigation and flap-\ncoverage instead of re-operation on cardiopulmonary bypass and explantation of the\ninfected prosthesis?\nVarious interventional and operative options were considered during multidisciplinary\ndiscussions. The cardiothoracic team, including a senior surgeon providing a second\n“Timely bespoke bacteriophage–antibiotic combination successfully treats refractory Pseudomonas aeruginosa\nmediastinitis and vascular graft infection.”\nopinion, concluded that conservative management was preferable. Following\nmediastinal washout and targeted antibiotic therapy, the patient demonstrated\nsignificant clinical improvement, maintained a reasonable quality of life, and expressed\nreluctance to undergo another major surgical procedure. As this was not the primary\nfocus of the manuscript, these details were not included in the main text.\n[Point 5]\nLine 195: “Definitive surgery to remove the composite vascular graft was not possible”.\nAgain, I agree with you that the patient is not operable, but the wording might not be\nprecise enough, since it is always possible.\nWe agree with the reviewer and modified the sentence to ““Definitive surgery to\nremove the composite vascular graft was technically challenging with high operative\nrisks and carried with it significant morbidity and mortality which was unacceptable to\nthe patient.”\n[Point 6]\nWhen and how was the last follow-up performed? Did you perform an FDG-PET/ CT\nto control tracer uptake and steer antimicrobial treatment?\nHer last follow-up was in April 2025. She remained clinically well on oral levofloxacin\nsuppression, with no recurrence of bacteraemia. A repeat PET-CT performed at that\ntime demonstrated further reduction in tracer uptake at the proximal ascending aorta,\nin the region of the aortic valve prosthesis. This image has been included in the revised\nFigure 2."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have submitted a thoughtful and comprehensive revision that directly addresses the concerns raised in my initial review. The revised manuscript now presents a well-organized, clearly written, and mechanistically grounded account of a complex Pseudomonas aeruginosa vascular graft infection managed with a personalized phage–antibiotic combination. The addition of in vitro time-kill synergy assays, biofilm dispersion experiments, and mutant data demonstrating the role of the MexAB–OprM efflux pump as a phage receptor, and more, meaningfully establish mechanistic plausibility for antibiotic re-sensitization and demonstrate that the phage component of therapy had a rational biological basis rather than being empirically applied.\nThe authors have also improved clarity throughout the manuscript. The narrative flow between clinical management, microbiological work-up, and mechanistic interpretation is coherent and easy to follow. The revised figures, particularly those illustrating the phage therapy workflow and the integrated clinical–laboratory process, are well designed and enhance readability. Importantly, the authors have moderated earlier overstatements about causality and now appropriately describe the contribution of phage therapy as plausible but lean toward being ultimately circumstantial.\nSome limitations inevitably remain with an n = 1 study. The assertion that phage therapy “restored” fluoroquinolone susceptibility, while mechanistically supported by the MexAB–OprM data, remains unconfirmed given the absence of post-treatment isolates. It may also be helpful to clarify that the detected phage DNA indicates presence rather than demonstrated replication. The discussion could be slightly condensed to maintain focus.", "reviewer_3": "Dear authors,\nThank you for addressing these points. I have no further comments."}, "author_response": ">>> We sincerely thank Reviewer #1 for their thoughtful and encouraging feedback. We\nappreciate the recognition of our efforts to address the initial concerns and to clarify the\nmechanistic basis for the phage–antibiotic combination therapy. Your comments regarding the\nmanuscript’s organization, clarity, and moderate interpretation of causality are invaluable to us,\nand we are grateful for your detailed review and constructive suggestions, which have greatly\nimproved the quality of our work.\nIn line 333- 334 of the main manuscript, the statement “Phage were detected in patient blood\nsamples at day 8 and day 15 of phage therapy by PCR using primers designed to discriminate\nbetween the three phages” indicates presence of phage rather than demonstrated replication.\nWe have condensed the discussion slightly further as well.\n>>> We would also like to thank Reviewer #3 for their constructive feedback and for taking the\ntime to review our manuscript. We appreciate your positive assessment and are grateful for your\nconsideration."}]

Decision letter