Methenamine as an Alternative Treatment of Neisseria gonorrhoeae Urethritis? An In Vitro and In Vivo Study in Galleria mellonella

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The main question addressed by the research is to assess the in vivo efficacy of methenamine hippurate against Neisseria gonorrhea.

It add to the subject area the possibility to have another treatment option compared with the standard therapies and to overcome the issue of drug resistance.

In the introduction at least one-two sentences about the clinical manifestation of the infection by Neisseria gonorrhoeae at the genital site or the anal site should be inserted.

 The standard therapy suggested for this infection by the international guidelines (CDC and IUSTI) should be described in order to introduce the concept of the drug resistance.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Neisseria gonorrhoeae has developed resistance to all existing antibiotic classes, creating an urgent need for new treatments. The study by Kanesaka et al suggests repurposing a 100-year-old antiseptic drug for therapy of gonococcal infection resistant to ceftriaxone, the drug of choice for treating gonorrhea. The methenamine testing was performed on clinical isolates of N. gonorrhoeae as well as on the non-common Galleria mellonella (wax moth larvae) infection model. The results indicate that methenamine may be recommended for further investigation in the advanced in vivo models as a promising antimicrobial agent for the therapy of gonorrhea. The manuscript is quite compact and concise.

I have a several questions for the authors that might make sense to reflect in the manuscript.

1. Galleria mellonella is an invertebrate model with fundamental physiological differences from mammals. It lacks an urinary system, so methenamine cannot undergo pH-dependent conversion to formaldehyde (core mechanism). Immune response and drug metabolism of G. mellonella are not comparable to humans, so efficacy in larvae does not predict human urethral efficacy.
2. Methenamine requires acidic urine (pH ≤ 5.5) to hydrolyze into formaldehyde. However, the study did not measure larval hemolymph pH and did not confirm formaldehyde generation in vivo. What is about testing under human urinary pH conditions? Moreover, human urinary pH varies (diet, medications, comorbidities) and efficacy may be inconsistent.
3. Pharmacokinetic (PK) data. Can the authors estimate methenamine/formaldehyde concentrations in larvae? Human PK data (urinary concentrations >500 mg/L, lines 44 and 166) are cited but not validated in the model.
4. Why do the authors test only one isolate (#19.598) in vivo despite the availability of 18 isolates with different resistance profiles? High-ceftriaxone-MIC strains (e.g., WHO X, MIC = 2 μg/mL) were also not evaluated in vivo.
5. The authors suggests methenamine could "preserve ceftriaxone" by reducing resistance risk but did not test synergy between methenamine and ceftriaxone even in their invertebrate model.

Minor comments.

Lines 52-53. ‘Two recent RCTs…’. RCT = Randomized Controlled Trials? Please decipher for the first time

Figure 1. Please, enlarge the chemical structure of methenamine.

Figure 2 and Figure 3 could have a higher resolution. The graininess of the text is conspicuous.

References. Please format the references according to the journal's requirements.

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This is a short and very straightforward paper describing methenamine-hippurate sensitivity of Neisseria gonorrhoeae. The experimental design was very simple- screen some isolates with different antimicrobial resistant profiles and examine dose dependency in a Galleria moth infection. The conclusions were that Neisseria gonorrhoeae has an MIC consistent with measurable levels excreted in human urine. The Galleria moth larvae model showed that methenamine-hippurate could reduce mortality when delivered at a dose of 400mg/kg.

There were three limitations to the study.

WHO P was used in the Galleria model of infection, but its MIC was not in Table 1. It needs to be stated the WHO P is ceftriaxone sensitive which is why it has been used in the Galleria model. If it has an MIC of 300 mg, why was the maximum dose in the Galleria model only delivered at 400 mg/kg? 

The dosing for the Galleria larvae showed no toxicity at 800mg/kg or 1200mg/kg and yet neither of these doses were tested in the larvae infected with gonococci. It would have been more rigorous to try the 800mg/kg dose in the infection model to demonstrate dose dependent effects. I think adding this condition into Figure 3 would make the paper much stronger. 

As I understand the paper, the concept is to use methenamine-hippurate against multi-drug resistant isolates. So I wondered why wasn't the ceftriaxone resistant strain WHO X isolate used in the Galleria moth larvae as this would prove the thesis that this treatment could be of clinical use.

Overall, this is a really interesting study and it could attract a lot more citations if the limitations above are addressed.

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The Percent match using the iThenticate report is 31%: please, reduce it.

Author Response

Reviewer 1:

The Percent match using the iThenticate report is 31%: please, reduce it.

 

Reply:

This has been done

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have addressed all comments and questions. The manuscript has undergone some changes, particularly in the Discussion section, which has become more engaging. The quality of the drawings has also improved where possible. The authors have provided clear and convincing responses to comments with which they disagree. Therefore, the manuscript is recommended for publication

Author Response

Thank you. We appreciate your comments and time.

Reviewer 3 Report

Comments and Suggestions for Authors

I have no further comments or suggestions.

Author Response

Thank you. We appreciate your comments and time.