In Vitro Activity of Finafloxacin against Panels of Respiratory Pathogens
Round 1
Reviewer 1 Report
In this article, Harding et al. show the microbicidal capacity of finafloxacin at different pH ranges against clinical isolates from patients with cystic fibrosis. I consider that the article could be of interest, however, there are two issues that it would be desirable for the authors to analyze or discuss before recommending it for publication.
- The authors performed the comparative analysis of the values ​​obtained for MIC50 and MIC 90 for finafloxacin and tobramycin. Although in general against various clinical isolates it is observed that finafloxacin is more efficient, they also observed that in some conditions this is not so clear for species commonly associated with chronic colonization in patients with cystic fibrosis such as Pseudomonas aeruginosa as well as Burkholderia cepacia. Therefore, it would be interesting if the authors were able to analyze the combined effectiveness of both antibiotics against these species.
- There are various reports mentioning the adverse effects of some fluoroquinolones (e.g., temafloxacin, trovafloxacin and grepafloxacin) due to their topoisomerase II inhibitory activity. Although it has been found that the topoisomerase II of prokaryotic cells is more sensitive to inhibition by quinolones than that of eukaryotic cells, it would be desirable for the authors to analyze the possibility of the appearance of damage in eukaryotic cells.
Author Response
- The authors performed the comparative analysis of the values ​​obtained for MIC50 and MIC 90 for finafloxacin and tobramycin. Although in general against various clinical isolates it is observed that finafloxacin is more efficient, they also observed that in some conditions this is not so clear for species commonly associated with chronic colonization in patients with cystic fibrosis such as Pseudomonas aeruginosa as well as Burkholderia cepacia. Therefore, it would be interesting if the authors were able to analyze the combined effectiveness of both antibiotics against these species.
Absolutely, this work was to requested investigate finafloxacin as an alternative to tobramycin for the treatment of respiratory infections, based on the superior efficacy of finafloxacin in conditions of low pH. The next set of experiments would be investigating the combined effectiveness of therapies, we are currently identifying a source of funding to enable us to do this. This is something we have shown before with finafloxacin and doxycycline against Burkholderia pseudomallei in vitro and in vivo (Barnes et al (2022) Investigation of a combination therapy approach for the treatment of melioidosis. Frontiers in Microbiology, Infectious Agents and Disease. 13: 934312).
- There are various reports mentioning the adverse effects of some fluoroquinolones (e.g., temafloxacin, trovafloxacin and grepafloxacin) due to their topoisomerase II inhibitory activity. Although it has been found that the topoisomerase II of prokaryotic cells is more sensitive to inhibition by quinolones than that of eukaryotic cells, it would be desirable for the authors to analyze the possibility of the appearance of damage in eukaryotic cells.
This is a very good point (but probably not in the scope of this paper). To answer your question MerLion have performed preclinical and clinical safety studies with good results. Finafloxacin had a several hundredfold greater cleavage activity with the bacterial than with the eukaryotic Type II DNA Topoisomerase enzymes. A series of detailed studies covering the topic of genotoxicity revealed that human exposures to finafloxacin, based on plasma AUC, (the most appropriate measure given the mode of action), will be significantly lower for the clinical use than the threshold which induced first effects, and therefore it was concluded that such exposures will not pose a genotoxic risk. Consequently, a range of preclinical and clinical safety studies have been completed with good results and no evidence of genotoxic effects. We (at Dstl) have also incubated J774 mouse macrophages with different concentrations of finafloxacin (up to 20 µg/mL) for 3 days. Even with the highest concentration there was no evidence of damage to the cells and there was no difference in viability compared to untreated cells
Reviewer 2 Report
Comments and Suggestions for Authors
In this manuscript, the authors evaluate the in vitro activity of finafloxacin against typical bacteria causing infections in CF patients. These bacteria are mostly isolated from respiratory samples. Overall, this is an interesting communication, well written and easy to understand. However, the subject matter of the article is not new, even though it provides interesting insights into the bacteria that are commonly isolated from patients with cystic fibrosis. In this regard, the authors fail to mention an important previous paper reporting increased low pH activity of finafloxacin against several bacteria (In Vitro Spectrum of Activity of Finafloxacin, a Novel, pH-Activated Fluoroquinolone, under Standard and Acidic Conditions, Stubbings, W. et al. Antimicrob Agents Chemother. 2011 Sep; 55(9): 4394-4397.). This and other papers also report on the possibility of using this antibiotic in patients with cystic fibrosis. The novelty of this communication should be better indicated by the authors. Other important points of criticism are 1) Why is finafloxacin less active than tobramycin against certain bacteria (e.g. Ps aeruginosa)?
2) Why did you not test for Staphylococcus aureus?
3) The antibiotic activity should be reported for each of the strains tested and not as an average value.
4) Have the strains tested been genotyped?
Minor editing of English language required
Author Response
In this manuscript, the authors evaluate the in vitro activity of finafloxacin against typical bacteria causing infections in CF patients. These bacteria are mostly isolated from respiratory samples. Overall, this is an interesting communication, well written and easy to understand. However, the subject matter of the article is not new, even though it provides interesting insights into the bacteria that are commonly isolated from patients with cystic fibrosis. In this regard, the authors fail to mention an important previous paper reporting increased low pH activity of finafloxacin against several bacteria (In Vitro Spectrum of Activity of Finafloxacin, a Novel, pH-Activated Fluoroquinolone, under Standard and Acidic Conditions, Stubbings, W. et al. Antimicrob Agents Chemother. 2011 Sep; 55(9): 4394-4397.). This and other papers also report on the possibility of using this antibiotic in patients with cystic fibrosis. The novelty of this communication should be better indicated by the authors.
I have added this manuscript and the following sentence. Such bacterial strains can be difficult to treat therefore the ability of finafloxacin to retain activity in conditions of low pH (eg following the establishment of a bacterial infection) may identify an alternative therapeutic for the treatment of such infections.
Other important points of criticism are
- Why is finafloxacin less active than tobramycin against certain bacteria (e.g. Ps aeruginosa)?
As explained in the publication, lung infection usually induces an acidic pH in the lung tissue, especially in patients with cystic fibrosis. Therefore we consider the acidic pH as clinically relevant. Finafloxacin shows good activity in this acidic environment against P. aeruginosa even though the panel of clinical isolates appears to have a limited susceptibility to fluoroquinolones in general as demonstrated by the elevated MICs at neutral pH. Finafloxacin was less active at pH 7 than tobramycin against P. aeruginosa but at pH 5 finafloxacin was more active than tobramycin (MIC90 of 0.5 µg/mL for finafloxacin and 2 µg/mL for tobramycin).
In general the change in pH appears to fundamentally change the properties of some bacteria which may well explain the different activities of antibiotics in both pH environments. Mozaheb et al (2023) describes how P. aeruginosa can develop an altered phenotype to tolerate a reduction in environmental pH. At the level of the bacterial envelope, modified lipid A composition and a reduction of the bacterial inner membrane permeability and fluidity are among the changes P. aeruginosa undergoes at a mildly low pH. Lin et al (2020) has shown that P. aeruginosa is highly adaptive to the CF-like acidic pH environment forming biofilms and inducing antibiotic resistance faster in acidic conditions. This reduced the activity of antibiotics including ciprofloxacin or tobramycin.
Ciprofloxacin and finafloxacin both target bacterial DNA bound gyrase and topoisomerase IV, stopping DNA replication, which results in the release of double stranded DNA breaks. However, when the potency for both drugs against the E.coli enzymes was evaluated in vitro, finafloxacin was shown to be a five-fold more potent inhibitor of E. coli gyrase and 20-fold more potent against topoisomerase IV than ciprofloxacin, exhibiting a comparatively high level of activity against both bacterial enzymes. Different roles of DNA gyrase and topoisomerase IV in bacterial cell division have been described in various studies and especially the potency of drugs to inhibit the bacterial DNA gyrase has been discussed as a crucial parameter for their activity against nondividing cells. Consistent with this hypothesis, finafloxacin has a potent in vitro DNA gyrase inhibition and it displays better activity against the slowly or nondividing persister cells and biofilms.
- Why did you not test for Staphylococcus aureus?
There is already a significant amount of information on the in vitro activity of finafloxacin against S. aureus that is published or is being written for publication. This work focussed primarily on members of the Bcc and other respiratory (non S. aureus) isolates.
The antibiotic activity should be reported for each of the strains tested and not as an average value.
Showing all the individual data for each bacterial strain (n=150) for tobramycin and finafloxacin at both pH values would be an enormous table and probably not warranted for a short communication. We have included the MIC ranges to enable the reader to see the whole range of activity for different strains and the MIC90s and 50s (as per other publications).
Have the strains tested been genotyped?
Genotypic data for these isolates is not available.
Round 2
Reviewer 1 Report
In the new version, the authors answer my questions with arguments based on previous research, which I believe could be of interest to other researchers in the field of study in the paper and not just remain in a response to the review.
Therefore, I consider that these should be added as part of the discussion in the form of future perspectives (for the study of the combined antibacterial effect of finafloxacin and tobramycin). In the same way, expanding the discussion with a paragraph dedicated to the evidence generated by the authors themselves as well as preclinical and clinical studies to mention that finafloxacin does not have a genotoxic effect on eukaryotic cells would help support the proposal about of the possible use of finafloxacin for the treatment of these infections.
Author Response
In the new version, the authors answer my questions with arguments based on previous research, which I believe could be of interest to other researchers in the field of study in the paper and not just remain in a response to the review.
Therefore, I consider that these should be added as part of the discussion in the form of future perspectives (for the study of the combined antibacterial effect of finafloxacin and tobramycin).
In addition, investigating the combined effectiveness of therapies, may further benefit in the treatment of infections caused by these pathogens. This is something we have previously demonstrated with finafloxacin and doxycycline against B. pseudomallei in vitro and in vivo (34).
In the same way, expanding the discussion with a paragraph dedicated to the evidence generated by the authors themselves as well as preclinical and clinical studies to mention that finafloxacin does not have a genotoxic effect on eukaryotic cells would help support the proposal about of the possible use of finafloxacin for the treatment of these infections.
Previous studies have demonstrated that this activity is antibacterial (due to greater cleavage activity with the bacterial rather than the eukaryotic Type II DNA topoisomerase enzymes) and not due to toxicity, demonstrated when finafloxacin was incubated with J774 mouse macrophages with no toxic effects observed (data, unpublished).
Reviewer 2 Report
The authors have addressed my concerns and improved the quality of the manuscript. Therefore, I recommend publication in Antibiotics
Author Response
not further comments to address
