Assessment of Bacteriophage Pharmacokinetic Parameters After Intra-Articular Delivery in a Rat Prosthetic Joint Infection Model
, Mohammad Javad Shariyate 2,3, Prateek Misra 3, Shubham Laiwala 3,4, Ara Nazarian 2,3,4,5,6,†and Edward Kenneth Rodriguez 2,3,5,†
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
The described study was aimed at pharmacokinetic assessment of phage therapy for prosthetic joint infections in a rat model. My very major concern relates to the fact that phage-antibiotic combination therapy for PJIs is an established method with efficiency at 78-95% shown in a number of clinical trials. Phages are applied and, probably, will be applied as a supplement to antibiotics due to ethical concerns and their inability to provide 100% infection clearance (Fedorov et al. 2023). Therefore, much of the stated relevance remains questionable.
“little is known about optimal phage dosing and the basic pharmacokinetic parameters of phages in vivo.”; “much dosing frequency needed to elicit a therapeutic response are unknown” (Line 84) – Please see Supplementary File S2 of Clarke et al. (2020) for clinical dosages and delivery regimes.
“Bacteriophage contents in wound fluid were 102 –104 PFU/mL within 4 ± 1 days after the onset of phage therapy; the concentration of bacteriophages administered daily to patients through drains was therapeutic and amounted to at least 105 PFU/mL” (Fedorov et al. 2023).
Given animal model biases in comparison to real human organisms, it is advisable that further progress in phage therapy pharmacology should be achieved through clinical studies (Nilsson 2019).
Still, the provided information may be useful if it can be connected to bacteriophage taxonomy. But, there is seemingly no published whole genome sequence of the phage vB_SepM_Alex.
Therefore, (1) the precise contribution of the study to the clinical science should be re-evaluated.
(2) An annotated genome sequence of vB_SepM_Alex should be deposited in GenBank. The accession number should be made available in the main text.
Minor:
In Abstract, give the first occurrence of the bacterial species name in full.
Line 23. Mention the precise dose of bacteriophage. Mention that vB_SepM_Alex is a bacteriophage
Remove mentioning of future animal studies (Lines 264, 309).
Clarke AL, De Soir S, Jones JD. The Safety and Efficacy of Phage Therapy for Bone and Joint Infections: A Systematic Review. Antibiotics (Basel). 2020 Nov 10;9(11):795. doi: 10.3390/antibiotics9110795. PMID: 33182795; PMCID: PMC7697170.
Fedorov E, Samokhin A, Kozlova Y, Kretien S, Sheraliev T, Morozova V, Tikunova N, Kiselev A, Pavlov V. Short-Term Outcomes of Phage-Antibiotic Combination Treatment in Adult Patients with Periprosthetic Hip Joint Infection. Viruses. 2023 Feb 10;15(2):499. doi: 10.3390/v15020499. PMID: 36851713; PMCID: PMC9964274.
Nilsson AS. Pharmacological limitations of phage therapy. Ups J Med Sci. 2019 Nov;124(4):218-227. doi: 10.1080/03009734.2019.1688433. Epub 2019 Nov 14. PMID: 31724901; PMCID: PMC6968538.
Author Response
Please see the attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsGENERAL COMMENTS:
The authors presented “Assessment of Bacteriophage Pharmacokinetic Parameters after Intraarticular Delivery in a Rat Prosthetic Joint Infection Model” which is trying to present pharmacokinetic parameters which will be helpful for future phage therapy in antibiotic resistant infection.
Overall, the article effectively summarizes the objectives, methods, and key findings of the study. The article is well-written. With a few minor additions and clarifications, it will effectively capture the attention and interest of readers in the scientific community.
I provide below a few comments and suggestions for more in-depth analysis that authors might want to consider.
Introduction:
§ The text discusses phages' ability to target specific bacteria and work well with antibiotics, but it doesn't explain why phages are especially good for treating PJIs, particularly their effectiveness against biofilms or bacteria that don't respond to multiple drugs.
§ The text notes that there is limited information about how phages behave in the body, but it would be more helpful to explain why this information is crucial for phage therapy in PJIs, such as the difficulties in getting phages to reach and stay in joint infections.
§ The hypothesis could be clearer by specifying which aspects of how phages behave in the body are being studied (such as phage biodistribution, elimination, and half-life) to give a more focused and scientific direction.
§ There is little discussion about how the information being collected will actually help improve phage therapy.
§ Some sentences are lengthy. Please break down into shorter sentences to improve clarity.
Materials and Methods:
§ If the phage vB_SepM_Alex has been previously characterized, please provide the GenBank accession number.
§ What method was used for the purification of the phage lysate? Kindly include a detailed protocol.
§ Was endotoxin level measured and removed from the phage lysate? If so, please provide the specific information on this process.
§ Did you check the bacteria count before phage administration? How did you calculate the MOI (multiplicity of infection) to select dose of 100µL of ~109PFU/mL of phage?
§ Please include an ethical statement within the main manuscript.
Results:
§ Phage and Bacterial counts: Please mention the detection thresholds for bacterial and phage counts in tissues.
§ Combining groups with and without infection for PK analysis might cause bias, as infection status can affect phage clearance and bioavailability from the body. Please justify this.
Discussion:
§ The study is consistent with previous research on phage half-lives, but it doesn't offer new ideas about how phages work in joint infections and please discuss on the results from other studies about how the immune system removes phages.
§ The study mentions that phages half-lives between infected and non-infected models, but doesn't explain why. Talking more about how the immune system reacts in these different situations would help to understand better.
§ The study reported that phages are quickly removed from the body, but doesn't provide direct proof, like checking for antibodies against phages. More discussion is needed on phages clearance and its possible mechanism particularly in the joint infection could make the study clearer.
§ The study reported a shorter persistence of phages, but doesn't suggested the doses and frequency of phage administration for future phage therapy. Please discuss in details.
Comments for author File: Comments.pdf
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for Authors
The report presented by Young et al., describes using an S. epidermidis isolate the therapeutic potential and pharmacokinetics of the phage, vB_SepM_Alex in a prosthetic rat model. With paucity of data regarding phage kinetics in infection models, this work would provide useful information that would drive phage therapy forward for prosthetic joint infection and other infection systems.
Please find below suggested comments to make this manuscript a better scientific read.
Major comments:
1. The results did not reveal any therapeutic advantage of administering the phage as no significant difference of bacterial recovery was observed in both PJI and PJI+phage groups, and rats within the PJI+phage treatment group had relatively higher bacterial counts as shown by the error bars (Figure 2). The bacterium was undetected after 24 h in the PJI group, showing the bacteria itself may not be stable after 24 h (Figure 2). In the same way with the phage counts, they seemed to decrease with time in both PJI+phage and sterile implant+phage groups, thus, reflecting lack of phage stability in the system (Figure 3, Lines 260-261). With the lack of reduction of bacterial numbers and decrease in phage counts in the phage-treated groups, it is not clear from the data if there was any interaction between the phage and bacteria and potentially what role the phage was playing here. Since the pharmacokinetic study was conducted in the context of phage therapy, it would be helpful to contextualise the work by providing a clear inference of the data in phage therapy context, dosing (only one phage concentration, and single administration was examined) or at least if there was any interaction between the phage and the bacteria to justify the work. Or else, just the phage within the model would be sufficient to generate the result shown on lines 26-28.
Minor comments:
1. Line 19- write full scientific name at first mention
2. Line 23- how is the phage titre in comparison to the bacterial inoculum? This is important as it is a pharmacokinetics study
3. Line 46-pls explain if this was with S. epidermidis too, and what future work is required to take this further to contextualise your work.
4. Line 61-62- It is important to say a bit about S. epidermidis and justification of why it was ideal model for this work. Ie, its relationship/importance to PJI
5. Line 78-was the phage lysate purified and buffer exchanged to a non-nutritive medium eg PBS? This should be considered in the control as the controls of PJI+phage and Sterile Implant+phage with the former having BHI-an animal based medium and the latter in PBS (Lines 99, 129, 133 etc).
6. Line 113-state reference re: ethical clearance for the work. Provide reference number etc from approval committee
7. Line 176_S. epi???
8. Line 218-‘No bacteria could be isolated from blood’- was this for all groups? Please see figure 2
9. Line 231- Table 1, can this be represented using a graph? too many numbers to process here.
10. Line 241- ‘Gram’
11. Line 243- gold standard would be to sequence them or conduct strain specific PCR and sequence amplicons to confirm. Former would help reveal any resistance to phage necessitating cocktail optimisation.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 3 Report
Comments and Suggestions for AuthorsAll comments were addressed adequately.
