Combined bacteriophage and antibiotic therapy for refractory peritoneal dialysis-related peritonitis caused by Klebsiella pneumoniae

Domain: neuroscience
Published: 2026-02-07
Editorial outcome: minor_revision

Abstract
Phage therapy is emerging as a promising alternative to antibiotics for treating various infections. However, there have been no prior studies on using bacteriophages for peritonitis in patients undergoing peritoneal dialysis. This report presents the successful treatment of refractory peritonitis in a 71-year-old male peritoneal dialysis patient using bacteriophages combined with antibiotics. The patient has a history of refractory and repeat peritonitis caused by Staphylococcus haemolyticus, which was resolved through simultaneous catheter replacement (SCR). Subsequently, the patient experiences another episode of refractory peritonitis due to Klebsiella pneumoniae. Although this strain is found to be susceptible to amikacin and imipenem, a 14-day course of treatment with these antibiotics in the abdominal cavity fails to resolve the peritonitis. Combined with antibiotic therapy, the patient is successfully treated with intraperitoneal phage therapy targeting his bacterial isolate. We monitor the longitudinal progression of phage loads, phage-neutralizing antibodies, interleukin-6 levels, and lipopolysaccharide concentrations in the dialysate effluent during the bacteriophage therapy. The combination of a phage cocktail and imipenem (IPM) demonstrates a greater effect in killing bacteria than either treatment alone, which indicates that a synergistic effect exists between the phage cocktail and IPM. Intraperitoneal IPM is discontinued after a 3-week course of treatment. At the same time, oral fluconazole is given to prevent fungal infections. The patient is discharged without any antibiotics. After this round of treatment, the patient remains healthy during the one-month follow-up. Our study suggests that personalized phage therapy combined with sensitive antibiotics can play a significant role in managing refractory peritonitis in patients undergoing peritoneal dialysis, showing promise for future applications.

Introduction
Bacteriophages, or phages, are viruses that specially infect bacteria, leading to bacterial death through a process called lytic replication. The therapeutic potential of lytic phages is being increasingly explored for treating difficult-to-manage infections1. Peritonitis is a major cause of treatment failure in patients undergoing peritoneal dialysis (PD). Over the past few decades, the incidence of PD-related peritonitis has significantly decreased, largely due to improvements in connection techniques and protocols for decolonizing Staphylococcus. These advancements have primarily reduced the rate of Gram-positive peritonitis, resulting in an increased proportion of Gram-negative peritonitis. It has been reported that the incidence rate and proportion of Gram-negative bacterial peritonitis have increased gradually in recent decades, and the relapse and recurrence rate for Gram-negative bacterial peritonitis was higher than that for other pathogens, with severe clinical manifestations and poor prognosis2. Retrospective studies have indicated that Gram-negative peritonitis has higher risks of catheter loss and mortality when compared with Gram-positive episodes3–7. Among these bacteria involved, Klebsiella pneumoniae has been identified as the second most common Gram-negative pathogen responsible for monomicrobial PD-related peritonitis. This pathogen can lead to serious complications, including increased morbidity, a higher risk of PD catheter removal, and a greater mortality rate in PD-related peritonitis8.

Relapsing or repeat peritonitis caused by coagulase-negative Staphylococcus might indicate that the PD catheter is colonized with biofilm and catheter removal should be considered. If the PD effluent becomes clear with antibiotic therapy, many patients might benefit from the simultaneous re-insertion of a new catheter during the same procedure, provided they are on antibiotic coverage. This approach could help avoid the need for temporary hemodialysis9. However, the practice of simultaneous catheter replacement (SCR) remains controversial in cases of refractory peritonitis in PD patients. Current evidence suggests that refractory peritonitis may be caused by bacterial adherence along the intraperitoneal portion of the catheter. There has been a reported case where refractory peritonitis caused by Pseudomonas aeruginosa was successfully treated with SCR after a partial response to antibiotics10.

Local production of interleukin-6 (IL-6) serves as a marker of ongoing intraperitoneal inflammation in patients undergoing PD11,12. The concentrations of IL-6 in drained PD fluid (PDF) are significantly higher than in plasma under stable conditions. Moreover, levels of IL-6 in the dialysate increase shortly before the onset of, during peritonitis, and several months after clinically successful treatment for peritonitis, suggesting its local production and reflecting an intraperitoneal inflammatory state13–15. Lipopolysaccharide (LPS), also known as endotoxin, is a key component of the outer membrane of Gram-negative bacteria. It plays an essential role in maintaining cell wall integrity, acting as a barrier against harmful compounds and potential antibiotics in the environment16. LPS is released from the outer membrane during bacterial cell death or cell division and growth17. It has been reported that increased effluent LPS level was a prompt marker of Gram-negative peritonitis in PD patients18–20. It is necessary to measure the levels of both IL-6 and LPS in dialysate effluent during and after phage administration.

In this study, we reported the innovative use of personalized bacteriophage therapy in combination with antibiotics for treating refractory peritonitis caused by Klebsiella pneumoniae in a patient undergoing peritoneal dialysis.

Results
Patient history
A 71-year-old male patient with end-stage renal disease due to membranous nephropathy began continuous ambulatory peritoneal dialysis (CAPD) in November 2015. The patient had a history of hypertension. Additionally, the patient experienced renal anemia, which was treated with erythropoietin. However, as the peritonitis progressed, the patient’s anemia worsened, leading to an increase in the dosing of erythropoietin. The patient also had secondary hyperparathyroidism, which was managed with cinacalcet. The parathyroid hormone level of this patient remained stable during that period. His medical history was significant for three episodes of peritonitis. In October 2023, the first case of peritonitis was reported, and the culture results were negative. In February 2024, a second episode of peritonitis occurred, which was identified as Staphylococcus epidermidis. By May 2024, a third episode of peritonitis took place, involving Staphylococcus haemolyticus. All three episodes of peritonitis were successfully treated with effective antibiotics. The patient received CAPD, which included four exchanges: three with 1.5% glucose and one overnight with 7.5% icodextrin, totaling a volume of 8 L. In early July 2024, the patient experienced another episode of peritonitis with cloudy peritoneal fluid and mild abdominal pain. Cultures from the PDF identified Staphylococcus haemolyticus, confirming a case of repeat peritonitis. Due to concerns about biofilm formation, an SCR was performed on July 17, 2024, once the PD effluent became clear. After the SCR, low-dose abdominal dialysis treatment began the next day while the patient remained in a supine position. We also continued administering intraperitoneal vancomycin for further treatment (Fig. 1).

Refractory peritonitis caused by Klebsiella pneumoniae
The patient developed diarrhea three days after the SCR, and the PD effluent became cloudy again. We performed another bacterial culture of the effluent and adjusted the anti-infection regimen to include the systemic administration of tigecycline (50 mg q12h), combined with oral fluconazole (100 mg daily) and intraperitoneal teicoplanin (15 mg/kg per 5 days). On July 26, 2024, Klebsiella pneumoniae was identified in the PD effluent culture, with no growth of Staphylococcus haemolyticus. The antibiotic sensitivity of the patient’s isolate of Klebsiella pneumoniae was presented in Table 1, which did not change over all the cultures obtained. Based on the antibiogram, the antibiotic therapy was switched to intraperitoneal amikacin (200 mg daily) and imipenem (500 mg daily), along with oral fluconazole (100 mg daily) to reduce the risk of fungal peritonitis. After 14 days of standard treatment, the PD fluid remained cloudy, and the white blood cell (WBC) counts in the PD effluent fluctuated from 1370 cells/μL to 11885 cells/μL. On August 1, 2024, Klebsiella pneumoniae was still present in the PD effluent culture. We considered this episode of peritonitis caused by Klebsiella pneumoniae as refractory, and according to the International Society for Peritoneal Dialysis (ISPD) guidelines, catheter removal should be considered. Due to the significant difficulty of attending the hemodialysis center, the patient was reluctant to switch from peritoneal dialysis to hemodialysis. In this case of refractory peritonitis, phage therapy might be a potential treatment option. Due to the ototoxicity linked with amikacin, it was discontinued after 12 days of treatment. The antibiotic regimen was adjusted to intraperitoneal imipenem, combined with oral fluconazole only on August 7, 2024. Despite the prolonged use of sensitive antibiotics, the patient experienced a slow but steady improvement, with a minimum WBC count in the effluent of 528 cells/μL (Fig. 1).

Intraperitoneal administration of bacteriophage
The treatment regimen consisted of a combination of antibiotics and included a 3-day phage therapy using the cocktail I (including phages ΦCP-p-KP-23240 and ΦCP-p-KP-21067) from August 13 to August 15, 2024, as described in the methods. Although the WBC counts in the PD effluent fluctuated, reaching up to 6296 cells/μL during the first round of phage therapy, Klebsiella pneumoniae strains were not detected after the completion of the phage therapy. On August 18, 2024, the WBC counts in the PD effluent sharply decreased to 26 cells/μL. The dynamic changes of plasma C-reactive protein (CRP) levels are shown in Fig. 1. By the end of the phage therapy, the plasma CRP level had dropped to a near-normal level. Intraperitoneal IPM and oral fluconazole were discontinued after a 3-week treatment course. While using the bacteriophage, this patient did not have any adverse events. The patient was discharged without any medicine on August 21, 2024 (Fig. 1). During the long-term follow-up, there was no relapse of Klebsiella pneumoniae peritonitis.

Biological characteristics of bacteriophages
The biological characteristics of ΦCP-p-KP-23240 and ΦCP-p-KP-21067 used in the therapy case need to be defined. Therefore, the genomes of ΦCP-p-KP-23240 and ΦCP-p-KP-21067 were extracted and sequenced. The results showed their genome sizes were 39766 bp and 38762 bp, respectively, indicating that they both belonged to the Podoviridae. Both genomes were circular double-stranded DNA and did not contain any endogenous genes, which indicated they were lytic phages. Besides, the multiplicity of infection (MOI) of ΦCP-p-KP-23240 and ΦCP-p-KP-21067 were respectively detected. The results showed they were correspondingly 10-5 and 10-6 (Fig. 2A and Fig. 2B), which indicated that both of them had extremely high lysis efficiency for the target bacteria. The one-step growth curve was determined at the MOI of 0.1, which was widely used and recognized. The results showed that ΦCP-p-KP-23240 and ΦCP-p-KP-21067 had a short latent period (0 min) and burst period (25 mins and 30 mins) (Fig. 2C and Fig. 2D), which was very beneficial to clinical therapeutics.

Synergistic effect for bacteriophages and antibiotics on clinical strains
The sensitivity test and the synergistic effect for bacteriophages and antibiotics were detected by the kill curve test. The result indicated that the bacteria of the patient could be killed by ΦCP-p-KP-23240 within 4 hours, followed by the production of phage resistance. By contrast, ΦCP-p-KP-21067 could not inhibit the growth of bacteria. However, the cocktail could make the resistant time extend to 24 hours or even longer (Fig. 3A), which indicated the phage-resistant mutants could be effectively cleared out by the ΦCP-p-KP-21067. Furthermore, the bacteria were sensitive to the 0.25 and 0.5 μg/mL IPM, but not 0.125 μg/mL (Fig. 3B), which was in accordance with the antibiotic sensitivity test results (Table 1). Moreover, the effect of killing bacteria by combining the utilization of phage cocktail and IPM was superior to the phage cocktail and 0.5 μg/mL IPM, as well as the others (Fig. 3C), which indicated that the synergistic killing effect existed between the phage cocktail and IPM.

Changes in dialysate IL-6 and LPS levels during phage therapy
Before the bacteriophage treatment began, dialysate IL-6 levels were maintained at a relatively high level, ranging from 22022.05 pg/mL to 42457.05 pg/mL. The levels of IL-6 in the PD effluent rose to 62659.55 pg/mL on August 14, 2024, the second day of phage therapy. The intraperitoneal IL-6 levels gradually declined after that, aligning with the changes in WBC counts in the dialysate effluent. After August 18, 2024, the IL-6 levels in the PD effluent remained relatively low (Fig. 4).

The levels of LPS in the PD effluent were monitored during the phage therapy. On August 3, 2024, a high concentration of LPS in the dialysate was recorded at 0.523 EU/mL before the initiation of phage therapy. Over the following days, the LPS levels gradually decreased, reaching a low of 0.007 EU/mL by August 7, 2024. During the phage treatment, there was a slight increase in the dialysate LPS levels, peaking at 0.123 EU/mL on August 16, 2024. This was followed by another decline, with levels dropping to 0.005 EU/mL by August 18, 2024. After this date, LPS in the PD effluent was no longer detectable (Fig. 4).

Neutralizing antibodies of phages in dialysate effluent before and after phage treatment
Phage treatment might fail due to the rapid production of neutralizing antibodies against phages by the host in response to the treatment. We collected dialysate effluent pre-phage day 7 and post-phage days 1, 3, 5, and 15, and tested it for neutralizing antibodies binding to both phages, with and without heating. Our findings indicated that complement inactivation in the dialysate effluent was not sufficient to impact phage activity. The analysis of dialysate effluent before and after treatment revealed no increase in dialysate-mediated neutralization of both ΦCP-p-KP-21067 and ΦCP-p-KP-23240 infections, suggesting that insufficient neutralizing antibodies were produced to affect phage activity (Fig. 5).

Determination of phage titre in dialysate effluent
We collected dialysate effluent pre-phage day 7 and post-phage days 1, 3, 5, and 15, and tested it for the presence of phage in dialysate effluent, both with and without heating. Our findings revealed that the phage load decreased by ~10,000-fold within one day after treatment and could not be detected after 48 h for both ΦCP-p-KP-21067 and ΦCP-p-KP-23240 (Fig. 6). After the treatment, the patient was discharged with improved symptoms of infection. One month later, the patient was readmitted to the hospital for further evaluation and remained in good health.

Review of published literature
After conducting extensive literature research, a total of 14 patients have received phage therapy for infections caused by Klebsiella pneumoniae. Out of these, 13 cases were successfully treated or showed clinical improvement, while 1 case did not respond to the treatment. The infection sites among these 14 patients included: 7 urinary tract infections, 2 post-fracture infections, 2 pulmonary infections, 2 post-joint replacement infections, and 1 case of purulent meningitis. Eleven patients received phage therapy in combination with antibiotics, while 3 patients received phage therapy alone. Throughout the phage therapy process for all 14 patients, no notable side effects were observed (Table 2).

Methods
Klebsiella pneumoniae clinical isolates and antibiotic susceptibility assay (AST)
Before phage therapy, a clinical strain of Klebsiella pneumoniae was isolated from the patient’s peritonitis dialysate on July 26, 2024. The strain was cultured on MacConkey Agar plates and subsequently identified using mass spectrometry with Bruker MALDI (Matrix-assisted laser desorption/ionization) Biotyper Rapid Microbial Identification System (Bruker Daltonik GmbH). It was also cultured in Luria-Bertani medium at 37°C52,53. To assess antibiotic sensitivity, the agar disk diffusion method was employed under the Clinical and Laboratory Standards Institute (CLSI) standard. The bacterial load was measured using Semi-Quantitative Clinical Cultures as previously described54. In brief, the culture plate was divided into four quadrants, and the peritonitis dialysate was streaked from the first quadrant to the last using an inoculation loop. After a 24-h incubation period, the number of quadrants exhibiting bacterial growth was evaluated. A result of ‘0’ was recorded if no growth of Klebsiella pneumoniae was observed on the plate; ‘1’ was noted if growth occurred only in the first quadrant; ‘2’ was reported if growth was observed in both the first and second quadrants.

Susceptibility testing of phage in the library by the phage spot test
Phages against the patient’s Klebsiella pneumoniae isolate, which was cultured on July 26, 2024, were screened from the Bacteriophage Library (CreatiPhage Biotechnology Co., Ltd, Shanghai, China.) using the method of spot test and the double agar overlay method55. The susceptibility of target bacteria to phages was measured by the efficiency of plating (EOP) assay as previously described56. Briefly, the bacteria were added to the 10 mL 0.5% LB medium. After mixing, the mixture was poured onto the 15 cm plate containing 1.5% LB medium, followed by waiting to solidify. 2 μL of each Klebsiella pneumoniae phage (a total of 250 different phages) was dropped onto the bacterial lawn, followed by waiting to dry. Finally, the plate was cultured at 37°C overnight. The appearance of clear plaques means that the phages were sensitive to the bacteria. The experiment was performed three times. For the first round of phage therapy, two Klebsiella pneumoniae phages, ΦCP-p-KP-23240 and ΦCP-p-KP-21067, were selected to form the cocktail I. The titer of phage ΦCP-p-KP-23240 against the pathogenic bacteria from the patient was found to be 5×108 PFU/mL. Phage ΦCP-p-KP-21067 served as a second-line anti-tolerance phage but did not exhibit a lytic effect on the pathogenic bacteria57, and its titer against the expanded host was measured at 1×108 PFU/mL.

Genome sequencing and bioinformatics analysis of phages
The genomes of ΦCP-p-KP-23240 and ΦCP-p-KP-21067 were extracted using a viral genome extraction kit (QIAamp MinElute Viral Spin Kit, Qiagen, Hilden, Germany) following the instructions. The genomes were sequenced at Sangon Biotech, Shanghai, using the Illumina HiSeq 2500 platform and assembled using SPAdes 3.15.5. Their genome sequences have been deposited in the GenBank in the National Center for Biotechnology Information (NCBI, Genbank NO. PX120905 and PX120903) (https://www.ncbi.nlm.nih.gov/).

Multiplicity of infection and one-step growth curve of phages
The host bacteria, Klebsiella pneumoniae, were cultured to the logarithmic growth phase (OD600nm = 0.6) and adjusted to ~1×108 CFU/mL. Different amounts of ΦCP-p-KP-23240 were then added according to the multiplicity of infection (MOI) ratios of 1, 0.1, 0.01, 0.001, 0.0001, 0.00001, and 0.000001. For ΦCP-p-KP-21067, the MOI ratios used were 10, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001, and 0.000001. The mixtures were cultured for 8 h at 37°C while shaking at 180 rpm. After incubation, the cultures were centrifuged at 8000×g for 5 min at 4°C and subsequently filtered. Phage titers were detected using the double-layer plate method. This experiment was conducted in triplicate. For further analysis, phages (at an MOI of 0.1) were added to 0.1 mL of Klebsiella pneumoniae culture (1×108 CFU/mL) during the logarithmic growth phase and incubated for 5 min at 37°C. The mixture was then centrifuged at 10,000×g for 5 min at 4°C. The pellet was resuspended in 10.0 mL of fresh LB liquid medium and incubated at 37°C with shaking at 180 rpm. Samples of 200 μL were taken every 5 min during the first 30 min, and then every 10 min until reaching a total of 60 min, ultimately extending the time to 90 min. Two samples were collected at each time point. One sample was immediately filtered through a 0.22 μm filter and diluted for phage titer detection using the double-layer plate method. The other sample was treated with 1% (v/v) chloroform for 30 min at room temperature to determine the titer58. This experiment was also repeated in triplicate.

Sensitivity test and synergistic effect for bacteriophages and antibiotics
The sensitivity of the bacteria in the patient to the bacteriophages and antibiotics was detected by the kill curve test, as well as the synergistic effect of bacteriophages and antibiotics. Briefly, 190 μL of LB medium was added to one well of a 96-well plate, which was followed by adding 10 μL of the bacterial culture in the logarithmic growth phase. After mixing, 1 μL of bacteriophages or a specific concentration of imipenem (IPM, final concentration of 0.125, 0.25, or 0.5 μg/mL) was added to it. The well that only added the bacteria was the negative control. After all the above, the 96-well plate was placed in the BioTek 800TS (America). The parameters were set as a total of 24 h of testing time at 37°C, containing a 20-min interval as the reading period, which was performed at OD600nm. The experiment was performed three times.

Bacteriophage propagation and administration
The preparation of the phage agent followed established protocols57. Phages were cultured on their original host, resulting in lysates with titers exceeding 1×109 PFU/mL. These lysates were then purified using a CIM® Anion Exchange Column QA (BIA Separations, Slovenia) according to the manufacturer’s instructions. The concentrate was dialyzed against a 0.9% sodium chloride physiological solution three times, with each dialysis lasting a minimum of three hours. Finally, the phage-containing solution was sterilized through a 0.22 μm filter, aliquoted, and packaged at the Good Manufacturing Practice (GMP) facility of Zhongshan Hospital of Fudan University, Shanghai, China. The phage cocktail I used (ΦCP-p-KP-23240 and ΦCP-p-KP-21067) was diluted in saline to a total volume of 10 mL, achieving concentrations of 5×108 PFU/mL for ΦCP-p-KP-23240 and 1×108 PFU/mL for ΦCP-p-KP-21067. This cocktail was diluted in peritoneal dialysate to a final volume of 1 L, achieving a concentration of 106 PFU/mL for each phage, which was administered through the PD catheter. The phage solution was retained in the intraperitoneal cavity for four hours and then drained out. The phage cocktail was injected twice a day for 3 consecutive days and two doses were administered successively. Simultaneously, the external exit of the PD catheter was moistened with a sterile gauze soaked in the phage solution for at least 20 min.

Phage neutralization assays
As previously described33, each PDF sample was diluted 1:10 in phage buffer, which consisted of 10 mM Tris-HCl (pH 7.5), 10 mM MgSO4, and 68 mM NaCl. The diluted samples were then incubated with either ΦCP-p-KP-23240 or ΦCP-p-KP-21067 at room temperature for 24 h. After incubation, 10-fold serial dilutions were performed, with 3 μL of each dilution placed onto top agar, followed by incubation at 37°C for 24 h. Additionally, some PDF samples were inactivated by heating at 56°C for 30 min to deactivate the complement. Each sample was tested in triplicate on each bacterial strain and the mean value and standard deviation were determined for each dialysate sample.

Determination of phage titre in dialysate effluent
Peritoneal dialysis effluent was collected from patients before and after phage treatment to determine the phage titre. The peritoneal dialysis fluid was then centrifuged at 8000×g for 2 min. The phage titre was measured using the EOP, as described previously56. A volume of 5 μl from serial 10-fold dilutions of the phage was spotted on double-layer agar plates, onto which the host bacteria, Klebsiella pneumoniae, had been spread in the upper soft agar. The number of plaques was counted after overnight incubation. Additionally, some peritoneal dialysis fluid samples were inactivated by heating at 56°C for 30 minutes to deactivate the complement.

Determination of interleukin-6 levels in dialysate effluent
The concentration of IL-6 in the overnight drained PDF was measured using an enzyme-linked immunosorbent assay (ELISA). All samples were analyzed simultaneously and in duplicate to minimize both intra- and inter-assay variations. The IL-6 concentration was determined using the Quantikine® kit from R&D Systems Inc. (Minneapolis, MN, USA). The detectable range specified by the manufacturer was from 3.1 to 300 pg/mL. For samples that exceeded the detection level, we diluted them appropriately before conducting the test. The IL-6 value was reported as the mean of the parallel readings for each specimen. WBC counts in PD effluent and serum CRP levels were monitored, respectively.

Determination of lipopolysaccharide levels in dialysate effluent
A commercial photometric Limulus Amebocyte Lysate (LAL) assay kit (Charles River Laboratories International, Inc., Guangdong, China) was utilized to measure LPS levels in the overnight drained PDF. The detectable range specified by the manufacturer was 0.005–2.500 EU/mL, with an upper normal limit for plasma samples set at 0.053 EU/mL. Effluent specimens were stored in pyrogen-free tubes and processed within four hours. According to the manufacturer’s instructions, one serving of the LAL assay was dissolved in 250 mL of reagent water. One hundred microliters of each effluent were diluted tenfold and then subsequently heated in a 75°C thermostat for ten minutes. After cooling to room temperature, 50 mL of specimen diluent was combined with 100 mL of the LAL solution in a reaction tube. Each specimen analysis included parallel measurements in two individual tubes, as well as two duplicate tubes containing 100 mL of reagent water as the negative controls, and two duplicate tubes containing 100 mL of quality control solution as the positive controls. All the tubes were incubated at 37°C for 75 min and readings were taken using the LKM series Kinetic Tube reader (Lab Kinetics Ltd, UK). The LPS value was reported as the mean of the parallel readings for each specimen.

Literature search
To summarize the application of phage therapy in related infections caused by Klebsiella pneumoniae, we searched the PubMed database for related articles from 1985 to 2024. An electronic search was performed using the keywords “Klebsiella pneumoniae” and “phage” or “bacteriophage”.

Ethics approval statement
This study protocol was reviewed and approved by the Ethics Committee of Zhongshan Hospital, approval number [B2024-041R], and the participant gave written informed consent, according to CARE guidelines and in compliance with the Declaration of Helsinki Ethical Principles. Consent to publish clinical information potentially identifying individuals was also obtained.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "I congratulate the Authors on this useful submission and contribution to the phage therapy field.\nWhat are the noteworthy results?\n-Novel delivery method of phage therapy (PD fluid) topical around PD opening.\nWill the work be of significance to the field and related fields? How does it compare to the established literature?\n-Yes, yet it is a single case and is discussion generating.\nDoes the work support the conclusions and claims, or is additional evidence needed?\n-the abscence of culture is helpful but low count culture technqiues would have been helpful. Additionally Culture and DNA (PCR) and Sequencying (metagenomics) results taken several times during the period.\nAre there any flaws in the data analysis, interpretation and conclusions? Do these prohibit publication or require revision?\nPotentially revision: Susceptilbity testing could use kill curve analysis, Plaque size could be of benefit, characterisation of the phage including the presenest of synergy (or at least not antagonism with the antibiotic). The endotoxin concentration of the phage was not clearly seen.\nIs the methodology sound? Does the work meet the expected standards in your field?\n-Better characterisation of the phages including their genomes, better characterisation of the Kleb spp isolate including its genome.\nIs there enough detail provided in the methods for the work to be reproduced?\n-Not for the literature search as the start date (could be as early as 1966) and data pull date are not clearly indicated.\n-Please see comments on susceptibility testing.\n-More appropriate and Indepth discussion on phage treatment with or without antibiotics including modern and international references. Appreciation of limitation of a pubmed versus a systematic or scoping review.", "reviewer_2": "This is an interesting article written by Yang et al using phage therapy in combination with Imipenem and Fluconazole for the treatment of refractory K. pneumoniae peritonitis. To my knowledge, this is the first case that describes phage therapy use in a peritoneal dialysis patient. For this article, I have several concerns (listed below) before considering this paper to be published in Nature Communications.\nMajor:\n• What was the legal basis for giving phage therapy? Is it according to compassionate use and some local regulatory framework? Or is it under the Helsinki Declaration, Paragraph 37?\n• It is hard to imagine that a patient with end-stage kidney disease, who has been on dialysis for 9 years, does not have any other comorbidities attributed to CKD? Did the patient have anemia (was it treated?), or did the patient not have secondary hyperparathyroidism?\n• What was the reason to treat the patient with no clinical improvement for 14 days in K. pneumoniae peritonitis episode? Is this following local guidelines?\n• There is no information given on phage morphology, genetic typing, and nature–lytic? Or lysogenic? This information is crucial for phage therapy.\n• There is no information/data given on phage interactions with antimicrobials used in combination for the treatment. This influences the microbiological outcome and clinical response as phages commonly have a synergistic effect with antibiotics but may also exhibit antagonism.\n• Why was only IgG tested? I would expect to see IgM and IgA curves as well. Additionally, the results indicate there were no preexisting phage-specific IgGs. Why did the authors test for neutralizing antibodies/reactions only in the PD fluid? Why not in serum? Demonstrating this would be essential to show the systemic effect of the immune response when administering phages intraperitoneally.\n• There is a lack of data on the proposed phage strategy. I would suggest creating infection curves using phages and then checking the susceptibility of phage-resistant mutants to antibiotics, as well as the susceptibility of the other phage used. Also, it would be essential to show the cocktail combination effect on phage resistance development.\n• I do not see the discussion part of SCR for S. haemolyticus peritonitis relevant for this paper, as it focuses on phage therapy of K. pneumoniae peritonitis.\nMinor:\n• In the title, please revise to indicate that the phages were given in combination with antibiotics, as this needs to be stated; otherwise, it is misleading to imply that the phages were used alone.\n• Line 39. In the abstract, please write how many days the patient was treated with Amikacin and Imipenem, instead of saying “prolonged”\n• In the abstract, please revise to include clinical outcome results.\n• In the introduction, lines 54-55, please add a reference showing the decrease of Gr+ and increase of Gr- peritonitis in PD patients.\n• Line 54, no need to add the word “bacteria”.\n• What is the reason to add a literature review on refractory and relapsing S. epidermidis treatment? As the article focuses on phage therapy use against refractory K. pneumoniae peritonitis?\n• For case report description, please follow CARE guidelines.\n• In the Methods section, Lines 97-98, did the patient have several K. pneumoniae strains? Or is this meant to be several isolates of the same strain?\n• Please add more detailed information on clinical isolates, including full antimicrobial susceptibility, minimum inhibitory concentration, and bacterial sequencing data.\n• In lines 119-121, please state clearly that two phages ΦCP-p-KP-23240 and ΦCP-p-KP-21067 were both used in the formed cocktail 2Φ, if I understand this correctly.\n• Is cocktail 2Φ the same as cocktail I?\n• Add information on the final phage concentration used for patient treatment. According to the written part in the Methods section (lines 122-124), it appears to be 10^6 PFU/ml.\n• In the methods section, include information on final product quality measures such as endotoxin level, microbiological burden, and pH.\n• Lines 128-130, please add more information on the phage susceptibility testing used; there is very limited or even no information given on this in the article.\n• For neutralization assays, phage susceptibility testing (EOP) and other methods were relevant, adding biological and technical replicate counts used in experiments.\n• In Lines 162-163, the authors state, “The LPS value was reported as the mean of the parallel readings for each specimen.” Was this the same for IL-6? Please be consistent, describing the methods and giving similar detail for the methods used.\n• In the patient history part, please add the dates (Month and Year) for previous episodes of peritonitis.\n• How was the first episode of S. haemolyticus peritonitis treated? What was the length of the antibiotic therapy?\n• Please specify the PD regimen the patient used when developing an episode of S. haemolyticus peritonitis. Was it CAPD or APD? What was the dialysis fluid volume and concentration used?\n• In line 186, on which day of treatment was the catheter changed?\n• In line 195, it is unclear whether the duration is measured in days after SCR or peritonitis treatment when the patient develops diarrhea.\n• Describe the phage spot test used in the case in the Methods section.\n• Which antibiotics were given to the patient in combination with phages? This is not mentioned in the text. In Figure 1, it states Imipenem only + antifungal Fluconazole. Were other antibiotics used as well?\n• Why were phages given only for three days?\n• Do you have longer follow-up data, as this happened already 11 months before? This would be important to know if the patient experienced relapse of K. pneumoniae peritonitis in the following year.\n• Please add information on adverse events, if any. In case no adverse events were present, please state this in the article.\n• Not knowing the endotoxin level in phage preparation, one can speculate that an increase in it comes from the phage preparation used.\n• I would suggest a biofilm assay for S. haemolyticus to prove the biofilm capability of the isolate; otherwise, it is only speculation, and there could be other reasons besides biofilm formation of bacteria.\n• In the discussion section, I would expect a more detailed discussion on the data obtained in the literature review and how it aligns with the present case. Additionally, a relevant comparison of phage application intraperitoneally (in animal models) should be provided, including reports on immune response.\n• I would suggest uploading data used for analysis into the repository for public availability and transparency."}, "author_response": "First of all, we would like to thank the reviewers for their time and constructive comments. We addressed their concerns, using Track Changes, in a revised manuscript. We feel that addressing the reviewers’ comments and questions improved the quality of our manuscript.\nPlease find below our point-by-point answers to the reviewers’ comments and questions.\nReviewer #1：\n• Does the work support the conclusions and claims, or is additional evidence needed?\nthe absence of culture is helpful but low count culture technqiues would have been helpful. Additionally, culture and DNA (PCR) and Sequencying (metagenomics) results taken several times during the period.\nResponse: Thank you for your advice. During the treatment process, we conducted multiple bacterial culture tests of the peritoneal dialysis fluid, but did not perform NGS testing. This was a limitation of our article, and we added “…while we conducted multiple bacterial culture tests on the peritoneal dialysis fluid during the treatment process, we did not perform metagenomic next-generation sequencing (mNGS). mNGS has been shown to improve the identification of pathogens in infectious diseases. Reports indicate that the pathogen detection rate of mNGS in the dialysis effluent of patients with peritonitis is significantly higher than that of traditional culture methods. By continuously monitoring the WBC counts in the peritoneal dialysis effluent and correlating them with the dialysate culture results, we can effectively evaluate the therapeutic outcome of peritonitis….” as one of the limitations in the second-to-last paragraph of the Discussion.\n• Are there any flaws in the data analysis, interpretation and conclusions? Do these prohibit publication or require revision?\nPotentially revision: Susceptibility testing could use kill curve analysis, Plaque size could be of benefit, characterization of the phage including the presents of synergy (or at least not antagonism with the antibiotic). The endotoxin concentration of the phage was not clearly seen.\nResponse: The kill curve analysis is essential for determining phage sensitivity and synergistic activity. We added “Sensitivity test and synergistic effect for bacteriophages and antibiotics” in the sixth paragraph of the Methods section. We also added “Synergistic effect for bacteriophages and antibiotics on clinical strains” in the fifth paragraph of the Results section. The experiments and comments are marked in yellow in the manuscript (as shown in Fig.3).\nThe endotoxin has extremely harmful effects for the health of the body. However, the bacteriophage preparations used in this phage therapy case were administered via peritoneal lavage instead of intravenous injection, which has few side effects according to the hundreds of therapy cases, e.g., a temporary increase in body temperature that can be decreased by physical measures. We also did not observe any internal endotoxin reactions, such as an increase in body temperature, in this patient after using the bacteriophage.\n• Is the methodology sound? Does the work meet the expected standards in your field?\nResponse: The bacteria of the patient in this case had been removed by the phage cocktail ΦCP-p-KP-23240 and ΦCP-p-KP-21067, combined with imipenem (IPM), which met the group-out criteria and the endpoint indicators and had an excellent therapeutic effect.\n• Better characterization of the phages including their genomes, better characterization of the Kleb spp isolate including its genome.\nResponse: Thanks for your valuable advice. The information about the characteristics of the bacteriophages was not written in the manuscript before. However, they are very important for phage therapy. Therefore, the biological characteristics of ΦCP-p-KP-23240 and ΦCP-p-KP-21067 are added in the manuscript, including the multiplicity of infection (MOI) and one-step growth curve (Fig.2). Moreover, their genome sequences have been deposited in the GenBank in the National Center for Biotechnology Information (NCBI, Genbank NO. PX120905 and PX120903). We added“Genome sequencing and bioinformatics analysis of phages” and “Multiplicity of infection and one-step growth curve of phages” in the Methods section. Furthermore, we added “Biological characteristics of bacteriophages” in the fourth paragraph of the Results section.\n• Is there enough detail provided in the methods for the work to be reproduced?\nNot for the literature search as the start date (could be as early as 1966) and data pull date are not clearly indicated.\nResponse: Sorry for the confusion, and thanks for your valuable advice. We added “…we searched the PubMed database for related articles from 1985 to 2024” in the last paragraph of the Methods section. Correction: After re-searching, a total of 14 patients who received treatment for refractory infections using bacteriophages were finally identified. Patient 3 was an updated case, who was a case of refractory urinary tract infection caused by Klebsiella pneumoniae using bacteriophage treatment (Ref. 72). We have added the relevant information about this patient in Table 2. Sorry again for this omission.\n• Please see comments on susceptibility testing.\nResponse: Thank you for your careful review. Indeed, our previous description is ambiguous and not exact. Only ΦCP-p-KP-23240 can lyse the target bacteria, but not the ΦCP-p-KP-21067, which can lyse the phage-resistant mutants. The sentence in the third paragraph of the Methods section “For the first round of phage therapy, we utilized two lytic phages, ΦCP-p-KP-23240 and ΦCP-p-KP-21067, both of which demonstrated efficient lytic activity against the Klebsiella pneumoniae strain, forming cocktail I.” is revised as follows: “For the first round of phage therapy, two K. pneumoniae phages, ΦCP-p-KP-23240 and ΦCP-p-KP-21067, were selected to form the cocktail I. The titer of phage ΦCP-p-KP-23240 against the pathogenic bacteria from the patient was found to be 5×10^8 PFU/mL. Phage ΦCP-p-KP-21067 served as a second-line anti-tolerance phage but did not exhibit a lytic effect on the pathogenic bacteria (Ref 26), and its titer against the expanded host was measured at 1×10^8 PFU/mL.”\n• More appropriate and indepth discussion on phage treatment with or without antibiotics including modern and international references. Appreciation of limitation of a pubmed versus a systematic or scoping review.\nResponse: Thank you for your valuable advice. We added more appropriate and in-depth discussion on phage treatment in the second to the fifth paragraphs of the Discussion section\nReviewer #2:\n• What was the legal basis for giving phage therapy? Is it according to compassionate use and some local regulatory framework? Or is it under the Helsinki Declaration, Paragraph 37?\nResponse: Yes. The informed consent form was signed before phage therapy according to compassionate use, which is under the Helsinki Declaration, Paragraph 37.\n• It is hard to imagine that a patient with end-stage kidney disease, who has been on dialysis for 9 years, does not have any other comorbidities attributed to CKD? Did the patient have anemia (was it treated?), or did the patient not have secondary hyperparathyroidism?\nResponse: Sorry for this confusion. Thank you for your careful and meticulous review. The patient has a history of hypertension. Due to peritonitis, his blood pressure became critically low, leading to the discontinuation of his antihypertensive medication. Additionally, the patient experienced renal anemia, which was treated with subcutaneous injections of erythropoietin at a dosage of 5,000 IU biweekly. However, as the peritonitis progressed, the patient's anemia worsened, with a hemoglobin level dropping to 80 g/L, and we increased the dosing of erythropoietin to 5,000 IU triweekly. Furthermore, the patient also had secondary hyperparathyroidism, which was managed with cinacalcet at a daily oral dose of 25 mg. Currently, the parathyroid hormone level measures 403 pg/mL, indicating a relatively stable condition. We added “Additionally, the patient experienced renal anemia, which was treated with erythropoietin. However, as the peritonitis progressed, the patient's anemia worsened, leading to an increase in the dosing of erythropoietin. The patient also had secondary hyperparathyroidism, which was managed with cinacalcet. The parathyroid hormone level of this patient remained stable during that period.” in the first paragraph of the Results section.\n• What was the reason to treat the patient with no clinical improvement for 14 days in K. pneumoniae peritonitis episode? Is this following local guidelines?\nResponse: Sorry for the confusion. Thank you for your careful review. The patient and his family strongly refused to undergo hemodialysis and requested to continue with peritoneal dialysis. Based on the results of the peritoneal dialysis fluid culture and drug sensitivity tests, we adjusted the antibiotic treatment plan by changing amikacin to imipenem. The patient's abdominal pain symptoms improved compared to before, and the white blood cell count in the peritoneal dialysis fluid decreased, although the reduction was gradual. Despite the continued presence of Klebsiella pneumoniae in the peritoneal dialysis fluid culture, we decided to pursue phage therapy in accordance with the patient's wishes. Ultimately, this case of refractory peritonitis was successfully cured.\n• There is no information given on phage morphology, genetic typing, and nature–lytic? Or lysogenic? This information is crucial for phage therapy.\nResponse: Thank you for your comprehensive consideration. Indeed, it is important. We added “Genome sequencing and bioinformatics analysis of phages” and “Multiplicity of infection and one-step growth curve of phages” in the Methods section. Moreover, we added “Biological characteristics of bacteriophages” in the Results section.\n• There is no information/data given on phage interactions with antimicrobials used in combination for the treatment. This influences the microbiological outcome and clinical response as phages commonly have a synergistic effect with antibiotics but may also exhibit antagonism.\nResponse: Thank you for your comprehensive consideration again. We added “Sensitivity test and synergistic effect for bacteriophages and antibiotics” in the Methods section. Moreover, we added “Synergistic effect for bacteriophages and antibiotics on clinical strains” in the Results section (Fig.3). We further added the relationship between bacteriophages and antibiotics in the third paragraph of the Discussion section.\n• Why was only IgG tested? I would expect to see IgM and IgA curves as well. Additionally, the results indicate there were no preexisting phage-specific IgGs. Why did the authors test for neutralizing antibodies/reactions only in the PD fluid? Why not in serum? Demonstrating this would be essential to show the systemic effect of the immune response when administering phages intraperitoneally.\nResponse: Thank you for your advice. In this therapy case, the bacteremia did not occur in the patient, and the infection occurred in the local peritoneal cavity. And also, the phage cocktail was used in the peritoneal cavity. Therefore, we want to know whether neutralizing antibodies are produced, which could decrease the therapeutic effect of the phage. Neutralizing antibodies in serum seem not to be so important for the activity of phage in the peritoneal cavity; in other words, they could not affect the clearance of bacteria by phage in the peritoneal cavity. Furthermore, the production of neutralizing antibodies needs more time, usually several days. Therefore, we think that the detection of neutralizing antibodies in the PD fluid has more important significance.\nIn fact, we did not specifically detect IgG. We used the dialysate effluent to complete the detection, which is a mixture of many Ig. However, though a large proportion of antibodies can bind to phage, they cannot close the activity site used to infect the bacteria, which can not affect the therapeutic effect of phage. In contrast, that can be affected by neutralizing antibodies. Therefore, to avoid ambiguity, the “IgG” should be corrected to “neutralizing antibodies” in the third-to-last paragraph of the Results section.\n• There is a lack of data on the proposed phage strategy. I would suggest creating infection curves using phages and then checking the susceptibility of phage-resistant mutants to antibiotics, as well as the susceptibility of the other phage used. Also, it would be essential to show the cocktail combination effect on phage resistance development.\nResponse: The advice is very interesting and helpful for the phage therapy strategy and clinical outcome of patients. In fact, we have built a big K. pneumoniae phage library, in which different K. pneumoniae phages can lyse different capsule types of K. pneumoniae. We have accumulated and known the match-up relationship between the bacteria and phage. In fact, the bacteria of the patient in this case can produce phage-resistance to ΦCP-p-KP-23240 within 4 hours and become the phage-resistant mutant (shown in Fig.3). We have detected the susceptibility of the other phage of a lot phage-resistant mutants (the original strains are isolated from different patients but have same capsule type), and these phage-resistant mutants can be lysed by ΦCP-p-KP-21067 (cannot lyse the original bacteria). The addition of ΦCP-p-KP-21067 in this cocktail can make the resistant time extend to 24 hours or even longer (shown in Fig.3).\n• I do not see the discussion part of SCR for S. haemolyticus peritonitis relevant for this paper, as it focuses on phage therapy of K. pneumoniae peritonitis.\nResponse: Thank you for your valuable advice. In this case, the patient underwent simultaneous catheter replacement (SCR), which successfully treated the repeat and refractory peritonitis caused by Staphylococcus haemolyticus. Following your recommendation, we shortened the description and discussion of SCR for this patient, providing a detailed account of the patient's condition and the complex treatment process.\n• In the title, please revise to indicate that the phages were given in combination with antibiotics, as this needs to be stated; otherwise, it is misleading to imply that the phages were used alone.\nResponse: Thank you for your advice. We revised the title of the article into “Effective Treatment of Refractory Peritonitis Caused by Klebsiella pneumoniae through Personalized Bacteriophage Therapy Combined with Antibiotics in a Peritoneal Dialysis Patient: Case Report and Literature Review.”\n• Line 39. In the abstract, please write how many days the patient was treated with Amikacin and Imipenem, instead of saying “prolonged”\nResponse: Thank you for your advice. We used “…a 14-day course of treatment…” instead of “prolonged” in the Abstract section accordingly.\n• In the abstract, please revise to include clinical outcome results.\nResponse: Thank you for your valuable advice. We added “Intraperitoneal imipenem (IPM) was discontinued after a 3-week course of treatment. At the same time, oral fluconazole was given to prevent fungal infections. The patient was discharged without any antibiotics. After this round of treatment, the patient remained healthy during the one-month follow-up.” in the Abstract section.\n• In the introduction, lines 54-55, please add a reference showing the decrease of Gr+ and increase of Gr- peritonitis in PD patients.\nResponse: Thank you for your valuable advice. We added “It has been reported that the incidence rate and proportion of Gram-negative bacterial peritonitis have increased gradually in recent decades, and the relapse and recurrence rate for Gram-negative bacterial peritonitis is higher than that for other pathogens, with severe clinical manifestations and poor prognosis (Ref. 2).” in the Introduction section.\n• Line 54, no need to add the word “bacteria”.\nResponse: Thank you for your valuable advice. We deleted the word “bacteria” accordingly.\n• What is the reason to add a literature review on refractory and relapsing S. epidermidis treatment? As the article focuses on phage therapy use against refractory K. pneumoniae peritonitis?\nResponse: Thank you again for your valuable advice. In this case, the patient underwent simultaneous catheter replacement (SCR), which successfully treated the repeat and refractory peritonitis caused by Staphylococcus haemolyticus. Following your recommendation, we shortened the description and discussion of SCR for this patient, providing a detailed account of the patient's condition and the complex treatment process.\n• For case report description, please follow CARE guidelines.\nResponse: Thank you for your valuable advice. Yes, it should follow CARE guidelines for case report description. This case is more complex than typical case reports. We have also summarized previous literature, presenting it as an article.\n• In the Methods section, Lines 97-98, did the patient have several K. pneumoniae strains? Or is this meant to be several isolates of the same strain?\nResponse: Sorry for the confusion. It means the same strain, so we revised it into “Before phage therapy, a clinical strain of Klebsiella pneumoniae was isolated from the patient's peritonitis dialysate on July 26, 2024” in the Methods section.\n• Please add more detailed information on clinical isolates, including full antimicrobial susceptibility, minimum inhibitory concentration, and bacterial sequencing data.\nResponse: Thank you for your valuable advice. The antibiotic sensitivity of the patient's isolate of Klebsiella pneumoniae was presented in Table 1. The genome sequence of this isolate has not been determined. However, based on the phage bank susceptibility profile, it might be inferred that this clinical isolate was a KL25 capsular strain.\n• In lines 119-121, please state clearly that two phages ΦCP-p-KP-23240 and ΦCP-p-KP-21067 were both used in the formed cocktail 2Φ, if I understand this correctly.\n• Is cocktail 2Φ the same as cocktail I?\nResponse: Sorry for your confusion. The cocktail 2Φ was the same as cocktail I, so we revised “cocktail 2Φ” into “cocktail I” accordingly. Sorry again for this error.\n• Add information on the final phage concentration used for patient treatment. According to the written part in the Methods section (lines 122-124), it appears to be 10^6 PFU/ml.\nResponse: Sorry for your confusion. Yes, the final phage concentration used for patient treatment was 10^6 PFU/ml. We added “This cocktail was diluted in peritoneal dialysate to a final volume of 1 L, achieving a concentration of 10^6 PFU/mL for each phage, which was administered through the PD catheter.” in the Methods section accordingly.\n• In the methods section, include information on final product quality measures such as endotoxin level, microbiological burden, and pH.\nResponse: Thank you for your suggestion. The titer of phage ΦCP-p-KP-23240 against the pathogenic bacteria from the patients was found to be 5×10^8 PFU/mL. Phage ΦCP-p-KP-21067 served as a second-line anti-tolerance phage but did not exhibit a lytic effect on the pathogenic bacteria, as shown in our previous article (Ref. 26). Its titer against the expanded host was measured at 1×10^8 PFU/mL. Additionally, no colonies grew on the blood agar plates incubated at 37°C for 48 hours. Phage production was purified through three rounds of dialysis with physiological saline, and the final product was also dissolved in physiological saline. These lysates were subsequently purified using a CIM® Anion Exchange Column QA (BIA Separations, Slovenia) in accordance with the manufacturer's instructions. However, the endotoxin level and pH were not measured.\n• Lines 128-130, please add more information on the phage susceptibility testing used; there is very limited or even no information given on this in the article.\nResponse: Thanks for your advice, and sorry for the confusion. We added “Susceptibility testing of phage in the library by the phage spot test” in the third paragraph of the Methods section to describe in detail.\n• For neutralization assays, phage susceptibility testing (EOP) and other methods were relevant, adding biological and technical replicate counts used in experiments.\nResponse: Thanks for your advice, and sorry for the confusion. Yes, the experiment was performed three times. We added “The experiment was performed three times” in the Methods section.\n• In Lines 162-163, the authors state, “The LPS value was reported as the mean of the parallel readings for each specimen.” Was this the same for IL-6? Please be consistent, describing the methods and giving similar detail for the methods used.\nResponse: Thanks for your valuable advice. We added “The detectable range specified by the manufacturer was from 3.1 to 300 pg/mL. For samples that exceed the detection level, we will dilute them appropriately before conducting the test. The IL-6 value was reported as the mean of the parallel readings for each specimen…” in the Methods section.\n• In the patient history part, please add the dates (Month and Year) for previous episodes of peritonitis.\nResponse: Thanks for your advice, and sorry for the confusion. We added “In October 2023, the first case of peritonitis was reported, and the culture results were negative. In February 2024, a second episode of peritonitis occurred, which was identified as Staphylococcus epidermidis. By May 2024, a third episode of peritonitis took place, involving Staphylococcus haemolyticus.” in the first paragraph of the Results section.\n• How was the first episode of S. haemolyticus peritonitis treated? What was the length of the antibiotic therapy?\nResponse: The initial case of S. haemolyticus peritonitis was treated with intraperitoneal vancomycin at a dosage of 15 mg/kg, administered five days a week for three weeks, resulting in a complete cure.\n• Please specify the PD regimen the patient used when developing an episode of S. haemolyticus peritonitis. Was it CAPD or APD? What was the dialysis fluid volume and concentration used?\nResponse: During this episode of peritonitis, the patient received continuous ambulatory peritoneal dialysis (CAPD), which included four exchanges: three with 1.5% glucose and one overnight with 7.5% icodextrin, totaling a volume of 8L. We added “The patient received CAPD, which included four exchanges: three with 1.5% glucose and one overnight with 7.5% icodextrin, totaling a volume of 8 L.” in the first paragraph of the Results section.\n• In line 186, on which day of treatment was the catheter changed?\nResponse: Sorry for the confusion. A catheter SCR procedure was performed on July 17, 2024, and we added “on July 17, 2024” in the first paragraph of the Results section accordingly.\n• In line 195, it is unclear whether the duration is measured in days after SCR or peritonitis treatment when the patient develops diarrhea.\nResponse: Sorry for the confusion. The duration was measured in days after SCR. We revised into “The patient developed diarrhea three days after SCR, and the PD effluent became cloudy again…” in the second paragraph of the Results section.\n• Describe the phage spot test used in the case in the Methods section.\nResponse: The comments have been revised in the manuscript and marked in red. We added“Susceptibility testing of phage in library by the phage spot test” in the third paragraph of the Methods section.\n• Which antibiotics were given to the patient in combination with phages? This is not mentioned in the text. In Figure 1, it states Imipenem only + antifungal Fluconazole. Were other antibiotics used as well?\nResponse: Sorry for the confusion. Due to the ototoxicity linked with amikacin, it was discontinued after 12 days of treatment. The antibiotic regimen was adjusted to intraperitoneal imipenem combined with oral fluconazole only. We added “Due to the ototoxicity linked with amikacin, it was discontinued after 12 days of treatment. The antibiotic regimen was adjusted to intraperitoneal imipenem, combined with oral fluconazole only.” in the second paragraph of the Results section.\n• Why were phages given only for three days?\nResponse: The bacteria in the human body can undergo gene mutation under the intense pressure of phage, so that they can survive in the war between bacteria and phage, which will result in the change of the sensitivity of phage, especially K. pneumoniae and P. aeruginosa. So, three days is a key time point used to adjust the treatment plan based on the sensitivity of the phage.\n• Do you have longer follow-up data, as this happened already 11 months before? This would be important to know if the patient experienced relapse of K. pneumoniae peritonitis in the following year.\nResponse: Yes. During the long-term follow-up, the patient did not have a relapse of Klebsiella pneumoniae peritonitis. We added “During the long-term follow-up, there was no relapse of Klebsiella pneumoniae peritonitis....” in the third paragraph of the Results section.\n• Please add information on adverse events, if any. In case no adverse events were present, please state this in the article.\nResponse: Thank you for your advice. During the use of the bacteriophage, this patient did not experience any adverse events. We added “While using the bacteriophage, this patient did not have any adverse events...” in the third paragraph of the Results section.\n• Not knowing the endotoxin level in phage preparation, one can speculate that an increase in it comes from the phage preparation used.\nResponse: Yes, it can be one reason for the increasing level of endotoxin. In addition, there will be a brief increase in the level of endotoxin in some patients, especially in patients with acute infections. It can be because the bacteriophages rapidly and massively kill the bacteria, thereby releasing a large amount of endotoxin, which is often accompanied by an increase in body temperature. As the number of bacteria decreased rapidly, the level of endotoxins also decreased significantly, which can be seen as the same result in this case.\n• I would suggest a biofilm assay for S. haemolyticus to prove the biofilm capability of the isolate; otherwise, it is only speculation, and there could be other reasons besides biofilm formation of bacteria.\nResponse: Thank you for your advice. In this study, we confirmed that the peritonitis caused by S. haemolyticus was a case of repeat peritonitis, which raised suspicion of biofilm formation. Therefore, a catheter SCR procedure was performed once the PD effluent became clear. Following the SCR, the culture results from the tip of the removed catheter confirmed the presence of S. haemolyticus, further supporting our suspicion of biofilm formation by this bacterium. We should have performed a biofilm assay for S. haemolyticus to demonstrate the biofilm capability of the isolate. Unfortunately, we did not conduct this test. We have included a statement in the manuscript acknowledging this limitation: “Finally, conducting a biofilm assay for S. haemolyticus would have strengthened our findings by demonstrating the isolate's biofilm-producing capability during the SCR process…” in the second-to-last paragraph of the Discussion section.\n• In the discussion section, I would expect a more detailed discussion on the data obtained in the literature review and how it aligns with the present case. Additionally, a relevant comparison of phage application intraperitoneally (in animal models) should be provided, including reports on immune response.\nResponse: Thank you for your valuable advice. We have expanded the relevant discussion section. We added “In previous overview of the first 100 consecutive real-world cases of personalized bacteriophage therapy treatment……” in the fifth paragraph of the Discussion section.\n• I would suggest uploading data used for analysis into the repository for public availability and transparency.\nResponse: Thank you for your suggestion. We have uploaded the source data for public availability and transparency."}, {"round": 2, "reviewer_comments": {"reviewer_2": "Before the publication, please consider adding minor revisions to your manuscript:\n1) Consider adding information on phage and imipenem synergy in the abstract.\n2) The reason the patient stayed on peritoneal dialysis. In your answer, you write that it was the patient's wish and he refused to change to hemodialysis. This would be important to add.\n3) In Fig. 1, please correct Klebsiella pneumoniae (it should be with the first letter in capital).\n4) In Table 1, please correct the spelling of Fosfomycin.\n5) In Figure 3. Please change the color for bacteria+ phages+0.5 mg/ml IPM, as it is difficult to see the result with the current color.\n6) In Table 2, please change the name “Dutch” to “the Netherlands”."}, "author_response": "I would like to express my gratitude to the reviewers for their time and valuable feedback. I am pleased to hear that our manuscript is, in principle, approved for publication. We have addressed their comments in a revised manuscript, which includes tracked changes.\nPlease find below our point-by-point answers to the reviewers’ comments and questions.\nReviewer #2：\n1) Consider adding information on phage and imipenem synergy in the abstract.\nResponse: Thank you for your advice. We added “The combination of a phage cocktail and imipenem (IPM) demonstrates a greater effect in killing bacteria than either treatment alone, which indicates that a synergistic effect exists between the phage cocktail and IPM.” in the Abstract.\n2) The reason the patient stayed on peritoneal dialysis. In your answer, you write that it was the patient's wish and he refused to change to hemodialysis. This would be important to add.\nResponse: Thank you for your advice. We added “Due to the significant difficulty of attending the hemodialysis center, the patient was reluctant to switch from peritoneal dialysis to hemodialysis.” in the second paragraph of the Results section.\n3) In Fig. 1, please correct Klebsiella pneumoniae (it should be with the first letter in capital).\nResponse: Thank you for your advice. We have corrected accordingly.\n4) In Table 1, please correct the spelling of Fosfomycin.\nResponse: Thank you for your advice. We have corrected accordingly.\n5) In Figure 3. Please change the color for bacteria+phages+0.5 mg/ml IPM, as it is difficult to see the result with the current color.\nResponse: Thank you for your advice. We have changed it accordingly.\n6) In Table 2, please change the name “Dutch” to “the Netherlands”.\nResponse: Thank you for your advice. We have corrected accordingly."}]

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