First Use of Phage Therapy in India for the Treatment of a Life-Threatening, Pan-Drug-Resistant Klebsiella pneumoniae Periprosthetic Joint Infection

Gopal Nath^1*, Alakh Narayan Singh¹, Gunjan Priyam², Swaroop Patel³, Govind Kumar Rai^4
1Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi-221005, India
²Department of Microbiology, Mahatma Gandhi Institute of Medical Sciences, Sewagram, Wardha, Maharastra-44200, India
³Orthopaedic Consultant, Apex Hospital, Bhikharipur, Varanasi -221004, India
⁴Biosafety Support Unit-DBT, Ground  Floor, Block-II, Technology Bhawan, Quatab Institutional Area, New Delhi 110016, India

*Correspondence author: Gopal Nath, Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi-221005, India; Email: [email protected]   

Citation: Nath G, et al. First Use of Phage Therapy in India for the Treatment of a Life-Threatening, Pan-Drug-Resistant Klebsiella pneumoniae Periprosthetic Joint Infection. J Clin Immunol Microbiol. 2025;6(2):1-16.

Copyright^© 2025 by Nath G, et al. All rights reserved. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Table 1: Outlines of the protocol for administering treatment regularly during the first 16 days and subsequently, along with the relevant laboratory findings, to assess the progress of the phage therapy.

Materials and Methods

Isolation of Bacteria from Clinical Specimens

The patient’s clinical samples (pus and urine) were inoculated on McConkey agar, blood agar and CLED (Cystine Lactose Electrolyte Deficient) media and incubated at 37°C overnight. The bacterial isolate was identified using standard microscopy and test substrates. The determination of antibiotic susceptibility for different isolates was assessed using the disk diffusion (Kirby-Bauer method) or broth microdilution (colistin) methods, following the guidelines of the Clinical and Laboratory Standards Institute (CLSI, 2020). 

Isolation of K. pneumoniae-Specific Bacteriophages

The K. pneumoniae (KpnBHU109) specific bacteriophages were isolated from the sewage drainage system of the University Hospital and Trauma Centre, BHU, Varanasi, using a slightly modified version of the previously described method [14]. Table 2 shows the accession number on NCBI and none of the phage genomes had antibiotic resistance or known virulence genes.

Bacteriophage Cocktail Against A. baumannii

We have a collection of 10 bacteriophages against A. baumannii isolates as an outcome of a completed research project. The phages were checked against the A. baumannii isolates and 3 actively lytic phages were selected for therapeutic purposes. The phages were purified and preserved for ready use. These phages were not sequenced.

Table 2: Bacteriophage genome data submitted to GenBank.

Results

We immediately provided a cocktail of 3 bacteriophages having broad-spectrum lytic activity against K. pneumoniae for empirical use on December 2, 2023. The pus sample was subjected to culture daily. The patient was shifted to the ICU to monitor any serious adverse effects. The phage cocktails were pushed inside the stitched wound through a catheter. The course of phage therapy is given in Table 1. The appearance of different bacterial species during phage therapy is shown in Fig. 1 and blood parameters are shown in Fig. 2.

Figure 1: The graph represents the appearance of K. pneumoniae, A. baumannii and Enterococcus faecium from the patient sample during the phage therapy.

Figure 2: The graph of blood parameters during the phage therapy. a) Total leucocyte count; b) C-reactive protein; c) Blood urea; d) Serum creatinine and e) Haemoglobin.

Discussion

Concerning the efficacy, safety and immune neutralisation of phage therapy, we undertook several in-vitro and preclinical studies. We carried out an in-vitro experiment on phage-antibiotic synergy, emphasizing its role in eradicating biofilms. The synergy is optimal when the antibiotic is administered 6-8 hours after the phage cocktails [15]. The first preclinical study involved inducing a wound on rat skin and establishing an infection with Pseudomonas aeruginosa. This infection was successfully treated using a bacteria-specific phage cocktail in an in-vivo wound model [16]. In the rabbit osteomyelitis model, acute and chronic Staphylococcus aureus-induced osteomyelitis were established by creating a crater near the distal end of the femur, which could be cured using a cocktail of seven phages. Both forms of osteomyelitis were successfully cured [17]. We further investigated the use of phage therapy to treat S. aureus-infected orthopaedic implants in a rabbit model, demonstrating the successful eradication of the bacteria [18]. Similarly, to address urinary tract infections, including chronic UTIs, we developed the model in mice and rats by inoculating the bacteria via the urethra to establish the infection, followed by determining the safe dose, dosage schedule and route for effective phage use. We conducted these preclinical studies on Escherichia coli, Klebsiella pneumoniae, Enterococcus faecalis and Enterobacter spp. A concentration of 109 PFU per dose administered through the urethra was the most effective, requiring only two doses to cure the infection [19,20].

In human studies, soft tissue-related chronic wound ulcers, we treated cohorts of patients, including a case series and a case-control study of phage cocktail. We successfully used customized phage cocktails for both diabetic and non-diabetic patients [21,22]. In a recent study, a double-blind, randomised controlled trial of phage therapy for chronically infected wounds resulted in a cure rate exceeding 92% [23]. Additionally, we conducted a case-control study using customised phage cocktails on acute, large, road traffic accident-contaminated wounds. We reported a one-third reduction in costs, length of hospital stays and healing without fibrosis, attributable to quicker recovery.  In this study, both groups underwent standard surgical therapy, while one group received additional treatment through the local application of customised phages [24].

We further conducted many preclinical studies to treat septicaemia in patients infected with MDR bacterial strains. The bacterial species involved in inducing septicemia in the mouse model encompassed all organisms in the ESKAPE group, namely Enterococcus species, S. aureus, A. baumannii, K. pneumoniae, P. aeruginosa and Enterobacter cloacae. We could decide the safe doses and route of administration of phage cocktail in such cases. It was found that for treating septicaemia, one should start with a minimal dose of phage cocktail, with continuous monitoring of the patient’s vitals (blood pressure, SpO2 and temperature rise) to avoid endotoxic shock due to sudden bacteriophage-induced bacterial lysis [14,18,25,26]. We also screened the phage therapy on a surrogate model of acute enteric fever (septicaemia) and chronic typhoid carriage, using a surrogate pathogen Salmonella Typhimurium. Additionally, we noted the successful eradication of chronic carriage [27].  We also searched for adverse reaction and toxicity studies. We observed that neutralising antibodies rise late, usually after three weeks of phage inoculation in rabbits [28]. We administered various concentrations of phages to several rats orally, ranging from very low to very high. It was intriguing to note that no adverse effects existed [29]. All these in-vitro, in-vivo (preclinical and clinical studies have been given in Table 3.

In-vitro Studies

In-vivo (Preclinical) Studies

In-vivo (Clinical) Studies

Table 3: Showing studies done at our centre before taking this first case of life-threatening septicaemia.

After conducting the aforementioned in-vitro, preclinical and clinical studies, we approached phage therapy with great caution.  We obtained the necessary consent from the patient and the treating physicians for a septicemic patient with a primary infection focus caused by pan-drug-resistant K. pneumoniae, associated with a hip implant wound. Phage therapy was initiated on compassionate grounds and the improvement was miraculous. On the 4^th day, after three doses of a specific phage cocktail, the discharge of frank pus ceased. The procalcitonin level decreased from 5.23 ng/mL to 0.298 ng/mL. The total leukocyte count was from 28740/µL to 15460/µL and the polymorph percentage was reduced from 87% to 72%. The other marker of acute inflammatory response, CRP, decreased from 240.1 mg/mL to 71.4mg/mL. The deteriorating kidney function showed a decrease in blood urea from 121 mg/dL to 70 mg/dL and a reduction in serum creatinine from 3.5 mg/dL to 1.0 mg/dL. It is worth noting that the bacteria were continuously detected for nearly 100 days during phage therapy. It indicates the longevity of the biofilm on the implant and no antibiotic can be used for such a prolonged period, even if it is found to be effective, without risking the development of resistance and adverse effects on the human microbiome [1,2,30]. The breaking down of the bacterial cell wall and EPS layer of the biofilm through the action of phage enzymes (glycan depolymerase, etc.) allows the diffusion of the bacteriophages, while the antibiotics cannot execute the same [31,32]. We can exploit this action of bacteriophages to use their synergy with antibiotics [33].

It was intriguing to note during therapy that the A. baumannii could be isolated when the CFU came down. It is quite likely that K. pneumoniae might have an inhibitory effect on A. baumannii in-vivo. It is worth mentioning that A. baumannii appeared twice. The second appearance of the bacterium occurred 31 days after the start of K. pneumoniae-specific phage therapy and a single course of the phage cocktail was likely inadequate for eradicating A. baumannii. This implies that in cases of mixed bacterial infections, phages targeting them should be administered for a sufficiently long duration if the disease is chronic.

All three phages used in the cocktail were lytic against the last time isolated bacteria. This observation implies that a cocktail of the phages prevents the development of mutants. Administering the phage cocktail at a high dose may induce neutralizing antibody formation. Fortunately, the antibody could be detected at the end of the therapy at a very low titre, i.e. <1:40 only. The reason might be that these phages are part of the natural phage biome of the body and colonized the patient during early childhood [28].

The patient was transferred to the ICU with the concern that a sudden release of bacterial endotoxins at high levels could lead to shock-like conditions. However, only a mild febrile reaction was observed after the first dose when the cocktail was administered. The toxin may be released in significant amounts with the first dose to induce fever; however, later on, due to a reduction in CFU, the amount of endotoxin released could be minimal. This finding suggests that localised infections may be treated with higher doses of the phage cocktail; however, in a severe septic state, the doses of the phage cocktail may need to be gradually increased [26]. Although many questions about the clinical efficacy and safety of bacteriophage therapy remain unanswered, particularly regarding cases involving Multidrug-Resistant (MDR) or Pan-Drug-Resistant (PDR) infections, several studies have already been conducted on safety, immunogenicity, dosing, routes of administration, treatment courses for specific infections, molecular characterization of each phage for clinical use, phage-antibiotic synergy and pharmacokinetics and pharmacodynamics. However, several carefully treated case reports are needed in clinical studies to support the safety and efficacy of phages and convince regulatory bodies to facilitate the large-scale application of phage therapy. This first Indian report treating life-threatening septicaemia bears great potential for phage therapy in chronic biofilm-associated infections with MDR/PDR in humans.

Conflict of Interest

The authors have declared no conflict of interest.

Data Availability Statement

The original contributions in the present study are included in the article/supplementary material; further inquiries can be directed to the corresponding author.

Funding

This work has no funding support.

Consent

Patient consent for phage therapy was taken.

Authors Contribution

GN: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Software, Supervision, Validation, Visualization, Writing – review & editing. ANS: Conceptualization, Data curation, Formal analysis, Investigation, Validation, Visualization, Writing – original draft, Writing – review & editing. GP: Formal analysis, Writing – original draft, Writing – review & editing. SP: Resource. GKR: Resource.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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