Effects of Lumacaftor-Ivacaftor on Airway Microbiota-Mycobiota and Inflammation in Patients with Cystic Fibrosis Appear To Be Linked to Pseudomonas aeruginosa Chronic Colonization

ABSTRACT Lumacaftor-ivacaftor is a cystic fibrosis transmembrane conductance regulator (CFTR) modulator combination approved for patients with cystic fibrosis (CF) who are homozygous for the F508del allele. This treatment showed significant clinical improvement; however, few studies have addressed the evolution of the airway microbiota-mycobiota and inflammation in patients receiving lumacaftor-ivacaftor treatment. Seventy-five patients with CF aged 12 years or older were enrolled at the initiation of lumacaftor-ivacaftor therapy. Among them, 41 had spontaneously produced sputa collected before and 6 months after treatment initiation. Airway microbiota and mycobiota analyses were performed via high-throughput sequencing. Airway inflammation was assessed by measuring the calprotectin levels in sputum; the microbial biomass was evaluated via quantitative PCR (qPCR). At baseline (n = 75), bacterial alpha-diversity was correlated with pulmonary function. After 6 months of lumacaftor-ivacaftor treatment, a significant improvement in the body mass index and a decreased number of intravenous antibiotic courses were noted. No significant changes in bacterial and fungal alpha- and beta-diversities, pathogen abundances, or calprotectin levels were observed. However, for patients not chronically colonized with Pseudomonas aeruginosa at treatment initiation, calprotectin levels were lower, and a significant increase in bacterial alpha-diversity was observed at 6 months. This study shows that the evolution of the airway microbiota-mycobiota in CF patients depends on the patient’s characteristics at lumacaftor-ivacaftor treatment initiation, notably chronic colonization with P. aeruginosa. IMPORTANCE The management of cystic fibrosis has been transformed recently by the advent of CFTR modulators, including lumacaftor-ivacaftor. However, the effects of such therapies on the airway ecosystem, particularly on the microbiota-mycobiota and local inflammation, which are involved in the evolution of pulmonary damage, are unclear. This multicenter study of the evolution of the microbiota under protein therapy supports the notion that CFTR modulators should be started as soon as possible, ideally before the patient is chronically colonized with P. aeruginosa. (This study has been registered at ClinicalTrials.gov under identifier NCT03565692).

Enaud and colleagues investigated how Lumacaftor-Ivacaftor, a novel combination therapy that restores function to the CFTR protein in cystic fibrosis patients, affects the composition of microbial communities and inflammation in the diseased CF lungs. The authors sampled patients prior to the initiation of Lumacaftor-Ivacaftor therapy and 6-months post-therapy and used nextgeneration sequencing technology to interrogate bacterial and fungal communities and biochemical laboratory tests to measure inflammatory markers.
Please see the attached PDF file for my major and minor review comments.
Reviewer #3 (Comments for the Author): In this paper the authors assess the effect of Lumacaftor-Ivacaftor on the lung microbiome in cystic fibrosis patients. The topic is of general interest and is well-written and clear to understand. This data has the potential to tell us about the overall microbiome alterations during disease progression under the treatment of targeted drugs such as LUM/IVA. Detailed notes below: -L79: italicize bacterial name -L88-89: grammar needs correcting -L126: A little more information would be helpful. How were these frozen? Liquid nitrogen? Straight into freezer? -20C or -80C? Others must be able to replicate your work exactly. -L148: Why was this kit chosen for DNA extraction? It is not recommended for fungal samples as it does not target the diverse cell structures of fungi. In fact, Qiagen created a DNeasy PowerSoil Pro kit specifically to assist with fungal isolations. DNA extraction techniques play a significant role in altering the composition of mycobiome studies. -L170/174: If ITS2 sequences were targeted for high throughput sequencing, why were 18S primers used to quantify fungal load? Why the switch between primer targets for fungi? -L215: These results are very interesting, but the overall effect is difficult to assess when negative controls are missing. For example, what about CF patients not receiving LUM/IVA? What about non-CF sputum samples? These controls would greatly increase the information that can be gleaned from this sequencing-based study.
-L342: Please expand on this topic. Aside from restating what was seen in the fungi, please address how this fits in the literature. Were these results expected? Why or why not? - Figure 1: some labels are blurry and need resolution fixed and could be bigger so readers can see the labels. Figure 1B in particular needs to be larger or better resolution.
- Figure 2E and 2F: P. aeruginosa is not italicized - Figure 3A, 3B, and S3B: The labels of the samples are not able to be read at all. Can these be made bigger? - Figure S2 axis labels need better resolution Staff Comments:

Preparing Revision Guidelines
To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the information as necessary. Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER. • Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file. • Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file. For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process. Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript. " Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me. If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
Enaud and colleagues investigated how Lumacaftor-Ivacaftor, a novel combination therapy that restores function to the CFTR protein in cystic fibrosis patients, affects the composition of microbial communities and inflammation in the diseased CF lungs. The authors sampled patients prior to the initiation of Lumacaftor-Ivacaftor therapy and 6-months post-therapy and used next-generation sequencing technology to interrogate bacterial and fungal communities and biochemical laboratory tests to measure inflammatory markers. I have a number of comments that I feel will help to enhance the quality of the manuscript as it was difficult to follow in parts.

Major comments
As a general comment, I would recommend that the authors consider reviewing the grammar used in the manuscript to ensure that the reader can follow along easily and that all valid points are highlighted appropriately.
Lines 105-109: There really needs to be a stronger literature review and summary of similar studies that have looked at the effects of CFTR modulator therapies on lung microbiota. You list a number of these in the References section (e.g. Graeber et al. 2021, Boutin et al. 2019Neerincx et al. 2021), but there should be some additional information in your introduction to really highlight the gap in knowledge in the field, the novelty of your study and its importance. What is currently known about the influence of CFTR correctors/modulators on lung microbiota? Are there significant shifts in diversity? Are they transient or prolonged? Is there an impact on the abundance of primary CF lung pathogens? What were issues with previous studies?
Line 122-123: I would appreciate some additional details on specimen collection here (or in an Appendix if necessary). Did any patients receive physical therapy prior to expectorating? Was the expectoration performed at home or in the clinic? Were they advised on how to collect the specimen? Was an oral rinse (e.g. saline mouthwash or gargle) used prior to specimen collection in order to evaluate the impact of oropharyngeal microbiota contamination of the expectorated sputa? How many days were there between sample collection and treatment initiation -please clarify if these were collected on the day of treatment?
Lines 214-222, 293: The authors highlight that there were both adolescent and adult patients included in the study, however no analyses account for this. I would also appreciate a deeper analysis based on disease stage -were there any disease related criteria used at the onset of study enrollment? Please comment in the methods (I recognize there are analyses based on FEV1 -please describe how the groups were defined). I would appreciate if there was some effort made to examine differences in adolescent and adult microbiota or those of individuals with early-moderate vs advanced disease given that we know that despite microbiota stability with disease progression there are fluctuations that may occur in adolescence/individuals with less severe pulmonary disease. Figure S2 is a good example of where this would be relevant.
Lines 260-263: The authors indicate that there was no significant difference in microbial diversity between pre-and mid-treatment specimens, including no changes in P. aeruginosa abundance, total bacterial or fungal loads among all patients. However, they claim in the same paragraph that the effects of the CFTR modulator therapy are driven by P. aeruginosa colonization (which is also indicated in the manuscript title). I do not feel there is sufficient evidence to support this claim, particularly as there were 34 patient samples (45% of the initial dataset -which is a limitation not mentioned in the Discussion) that were not included in the mid-treatment M6 dataset as they were unable to produce sputum. I would recommend that the authors revisit this portion of the manuscript and clarify their statements (e.g. change the wording to reflect a non-P. aeruginosa dominated microbiome). There is also a mention that shifts in alpha diversity were noted among those patients that did not have P. aeruginosa isolated. Please comment (in the discussion) as to whether increased diversity is anticipated to be beneficial overall to patients (either hypothetically or with literature support) given what is known about the CF lung microbiome and disease state (e.g. perhaps changes in diversity are seen with P. aeruginosa more at the strain level vs an overall shift in abundance). Why might this not have been observed for patients with P. aeruginosa? Were the study patients still receiving their standard CF antimicrobial therapeutic regimens in addition to the modulator therapies? Please comment on this as it may also influence some of the data seen if so.
Line 282: Can the authors provide justification for the 6 month sampling time point and a lack of any additional sampling past this point?

Minor comments
Lines 88-95: Please consider revising this section as it does not clearly articulate the point you are trying to make. What factors drive the evolution of disease and shape the CF lung microbiota? How would these be different to what CFTR modulators may achieve?
Lines 96-97: Please comment on "traditional" therapeutic regimens that were administered pre-CFTR modulators as this will help to contextualize the importance of modulator therapy. How common is the F508del mutation (Please state this in the text)? I would also recommend commenting on the length of Lumacaftor-Ivacaftor therapy (i.e. are they short-term or chronic therapies).
Line 118, 133: What clinical information was collected from your study patients? I would recommend commenting on this in the text.
Lines 136-143: What about other pathogens? In Tables 1 and 2 you separate these out into "pulmonary colonization" and "pulmonary chronic colonization". Can you elaborate on how you distinguished these categories?
Lines 152-157; Lines 160-161: Please provide additional information regarding the sequencing preparation (e.g. paired end reads, amplicon size, other parameters) and analysis in an Appendix as it is not reproducible in its current state. What were your quality metrics -please state.
Lines 170-172: This may be better suited in the Results section along with a summary of the sequencing quality metrics Lines 176-177: What concentration ranges were used here? Please comment on this.
Lines 181-187: Were the manufacturer's recommended protocols used? What controls were used and were any of the tests repeated? Were the positive Galactomannan results repeated in duplicate?
Lines 204-205: Were there any statistical corrections for multiple comparisons?
Lines 207-208: I would recommend having these codes available in GitHub or a similar online repository Line 223: Can the authors revisit the manuscript and be consistent with the order of bacterial and fungal analyses throughout the text? I have highlighted this line as an instance where it switches from fungalbacterial whereas in other parts of the text the analyses are mentioned bacteria-fungal.
Lines 231-241: Was there a difference in Gram positive vs Gram negative organisms with respect to abundance pre-or during therapy?
Line 246 -I would avoid using the term "uncolonized" and would prefer "not detected" or something similar, as it may be that it is at a level that is too low to detect by current methods Line 248-249: Please tone down the wording in this comment -as it is not necessarily true, this is only a measure at baseline Line 303, 368: Please use terms other than "morbi-mortality" and "a fortiori" Line 570: Table 1 is confusing to me on first glance and is difficult to interpret. It is unclear to me which of the columns "Missing Values" refers to -is it all of the columns? Similarly, you list "all patients N=75" etc in the other columns despite there being missing data. Please clarify. Instead of Table 1   Thank you also for the editorial and reviewer comments. We have revised the manuscript to address these comments. The corresponding modifications are visible using the "Track Changes" function in Microsoft Word and we added some comments. We would like to respond to the comments in detail as follows.

Reviewer 2 Comments:
Thank you for taking time to review the article. Please find our point-by-point answers.

Major comments
1. As a general comment, I would recommend that the authors consider reviewing the grammar used in the manuscript to ensure that the reader can follow along easily and that all valid points are highlighted appropriately.
We have revised the grammar of the manuscript in the hope that it will be easier to follow. but there should be some additional information in your introduction to really highlight the gap in knowledge in the field, the novelty of your study and its importance. What is currently known about the influence of CFTR correctors/modulators on lung microbiota? Are there significant shifts in diversity? Are they transient or prolonged? Is there an impact on the abundance of primary CF lung pathogens? What were issues with previous studies?
We have added a paragraph to the introduction to better situate the context of this study (see lines 113-

133)
3. Line 122-123: I would appreciate some additional details on specimen collection here (or in an Appendix if necessary). Did any patients receive physical therapy prior to expectorating? Was the expectoration performed at home or in the clinic? Were they advised on how to collect the specimen? Was an oral rinse (e.g. saline mouthwash or gargle) used prior to specimen collection in order to evaluate the impact of oropharyngeal microbiota contamination of the expectorated sputa? How many days were there between sample collection and treatment initiation -please clarify if these were collected on the day of treatment?
We have added these details in the material and method (see lines 148-152).
4. Lines 214-222, 293: The authors highlight that there were both adolescent and adult patients included in the study, however no analyses account for this. I would also appreciate a deeper analysis based on disease stage -were there any disease related criteria used at the onset of study enrollment? Please comment in the methods (I recognize there are analyses based on FEV1 -please describe how the groups were defined). I would appreciate if there was some effort made to examine differences in adolescent and adult microbiota or those of individuals with early-moderate vs advanced disease given that we know that despite microbiota stability with disease progression there are fluctuations that may occur in adolescence/individuals with less severe pulmonary disease. Figure S2 is a good example of where this would be relevant.
We have added the analyses at inclusion and for the evolution according to age in the results section, knowing that we have kept the choice to focus more on lung function than on age as a marker of disease severity (see lines 264-267; 281-283 and Figure S3) 5. Lines 260-263: The authors indicate that there was no significant difference in microbial diversity between pre-and mid-treatment specimens, including no changes in P. aeruginosa abundance, total bacterial or fungal loads among all patients. However, they claim in the same paragraph that the effects of the CFTR modulator therapy are driven by P. aeruginosa colonization (which is also indicated in the manuscript title). I do not feel there is sufficient evidence to support this claim, particularly as there were 34 patient samples (45% of the initial dataset -which is a limitation not mentioned in the Discussion) that were not included in the mid-treatment M6 dataset as they were unable to produce sputum. I would recommend that the authors revisit this portion of the manuscript and clarify their statements (e.g. change the wording to reflect a non-P. aeruginosa dominated microbiome). There is also a mention that shifts in alpha diversity were noted among those patients that did not have P. aeruginosa isolated. Please comment (in the discussion) as to whether increased diversity is anticipated to be beneficial overall to patients (either hypothetically or with literature support) given what is known about the CF lung microbiome and disease state (e.g. perhaps changes in diversity are seen with P. aeruginosa more at the strain level vs an overall shift in abundance). Why might this not have been observed for patients with P. aeruginosa? Were the study patients still receiving their standard CF antimicrobial therapeutic regimens in addition to the modulator therapies? Please comment on this as it may also influence some of the data seen if so.
As recommended, the authors revisit the title and clarify their statements, as follow: -The title has been modified as "Lumacaftor-Ivacaftor effect on CF lung microbiota-mycobiota and inflammation appears to be linked to Pseudomonas aeruginosa chronic colonization" -lines 295-297: section title has been changed as followed: "Changes in clinical outcomes, microbiotamycobiota and inflammation characteristics under lumacaftor-ivacaftor treatment appears to be linked to P. aeruginosa chronic colonization status at baseline" -lines 333-334: Discussion statements have been clarified.
-Regarding patients chronically colonized with P. aeruginosa, the authors have added comments in the discussion section (see lines 371-373; 414-416) as recommended.
6. Line 282: Can the authors provide justification for the 6 month sampling time point and a lack of any additional sampling past this point?
We discussed this point in the discussion section (see lines 418-426).

Minor comments
7. Lines 88-95: Please consider revising this section as it does not clearly articulate the point you are trying to make. What factors drive the evolution of disease and shape the CF lung microbiota? How would these be different to what CFTR modulators may achieve?
We have reviewed this section and further detailed the mechanisms of dysbiosis that may be involved in cystic fibrosis (see lines 88-91).
8. Lines 96-97: Please comment on "traditional" therapeutic regimens that were administered pre-CFTR modulators as this will help to contextualize the importance of modulator therapy. How common is the F508del mutation (Please state this in the text)? I would also recommend commenting on the length of Lumacaftor-Ivacaftor therapy (i.e. are they short-term or chronic therapies).
We have included all these details in the text (see lines 98-100 and line 102).
9. Line 118, 133: What clinical information was collected from your study patients? I would recommend commenting on this in the text.
We have included all these details in the text (see lines 160-161).
10. Lines 136-143: What about other pathogens? In Tables 1 and 2 you separate these out into "pulmonary colonization" and "pulmonary chronic colonization". Can you elaborate on how you distinguished these categories?
These colonization criteria were recorded by the physician during the follow-up of each CF patients. 11. Lines 152-157; Lines 160-161: Please provide additional information regarding the sequencing preparation (e.g. paired end reads, amplicon size, other parameters) and analysis in an Appendix as it is not reproducible in its current state. What were your quality metrics -please state.
The authors have provided additional information and stated on quality metric as follow: PCR products were checked on Agilent automated system (see lines 176, 182-183). As recommended in DADA2, we used standard filtering parameters [i.e. maxN=0 (DADA2 requires no ambiguous bases (Ns)), truncQ=2, rm.phix=TRUE and maxEE=2]. Of note, the maxEE parameter sets the maximum number of "expected errors" allowed in a read, which is a better filter than simply averaging quality scores.
Of note: DADA2 incorporates quality information into its error model which makes the algorithm robust to lower quality sequence.
12. Lines 170-172: This may be better suited in the Results section along with a summary of the sequencing quality metrics As recommended, the authors moved these data to the results section (see lines 190-191).
13. Lines 176-177: What concentration ranges were used here? Please comment on this.
We have specified the concentration ranges in the text (see lines 210-212).
14. Lines 181-187: Were the manufacturer's recommended protocols used? What controls were used and were any of the tests repeated? Were the positive Galactomannan results repeated in duplicate?
The We have homogenized this point in the manuscript.
18. Lines 231-241: Was there a difference in Gram positive vs Gram negative organisms with respect to abundance pre-or during therapy?
The authors compared relative abundances of bacterial ASVs with respect to pre-or during therapy; 10 bacterial species belonging to 4 Gram positive genus and 4 Gram negative genus expressed significant differences in relative abundances using DeSeq2 paired to species (see Table above). 19. Line 246 -I would avoid using the term "uncolonized" and would prefer "not detected" or something similar, as it may be that it is at a level that is too low to detect by current methods According to the new material & methods, the authors would prefer to modify this spelling and nuanced it by clearly referring to patients not chronically colonized with P. aeruginosa (instead of uncolonized patients).
20. Line 248-249: Please tone down the wording in this comment -as it is not necessarily true, this is only a measure at baseline We have nuanced this point (see lines 290-291).
21. Line 303, 368: Please use terms other than "morbi-mortality" and "a fortiori" We have changed these terms.
22. Line 570: Table 1 is confusing to me on first glance and is difficult to interpret. It is unclear to me which of the columns "Missing Values" refers to -is it all of the columns? Similarly, you list "all patients N=75" etc in the other columns despite there being missing data. Please clarify. Instead of Table 1 having a Missing values column, perhaps it would be better to have a "Number of patients with corresponding clinical data (N=)" column or something similar.
To avoid any confusion, we have retained in Table 1 only the clinical characteristics of all included patients and have added a table in supplementary data (Table S1) for the clinical characteristicsof the subgroups (with and without sputum at M6).
23. Line 629: Please add a breakdown of adolescent vs adult in Figure S1 As these were not subgroups defined a priori in the study design for the analysis, we proposed not to include this information in the flow chart but specified it in the text and in Tables 1 and S1.

Reviewer #3
Thank you for taking time to review the article. Please find our point-by-point answers.

L79: italicize bacterial name
We have corrected this point.

L88-89: grammar needs correcting
We have corrected this point.
3. L126: A little more information would be helpful. How were these frozen? Liquid nitrogen? Straight into freezer? -20C or -80C? Others must be able to replicate your work exactly.
We have indicated the procedure for storing the samples (-20°C) (see line 153).
4. L148: Why was this kit chosen for DNA extraction? It is not recommended for fungal samples as it does not target the diverse cell structures of fungi. In fact, Qiagen created a DNeasy PowerSoil Pro kit specifically to assist with fungal isolations. DNA extraction techniques play a significant role in altering the composition of mycobiome studies.
One of the reasons for choosing this kit is that we are also studying the intestine-lung axis, and therefore we needed to have a kit common to both ecosystems, in order not to induce extraction biases. We first checked with fabricant that the PowerFecal Pro was also efficient for the lysis of fungi and we were also able to verify with the mocks that our process allowed to find the different expected fungal species. As this FungiQuant has a limit of quantification 25 copies and a limit of detection at 5 copies, we chosen to use it to quantify efficiently fungal loads in the present project.
Since lumacaftor-ivacaftor treatment was part of standard care for deltaF508 homozygous patients older than 12 years, it was not ethically conceivable to have control patients with cystic fibrosis paired on age, sex, and mutations. However, longitudinal analysis of the respiratory microbiota allowed the patient to be taken for his own control. The authors have discussed this point in the discussion section.
7. L342: Please expand on this topic. Aside from restating what was seen in the fungi, please address how this fits in the literature. Were these results expected? Why or why not?
The authors have discussed this point lines 392-395.
8. Figure 1: some labels are blurry and need resolution fixed and could be bigger so readers can see the labels. Figure 1B in particular needs to be larger or better resolution.
We have made these changes 9. Figure 2E and 2F: P. aeruginosa is not italicized We have made these changes 10. Figure 3A, 3B, and S3B: The labels of the samples are not able to be read at all. Can these be made bigger?
We improved the resolution of the axes and removed the names of the samples that were not essential and overloaded the figure.
11. Figure S2 axis labels need better resolution We have improved the resolution of figure S2.
We hope that this revised version will now be found suitable for publication in Microbiology Spectrum.
Best regards, Thank you for submitting your manuscript to Microbiology Spectrum. As you will see your paper is very close to acceptance. As you will notice, one reviewer recommends improving clarity and removing the replication of data among Table 1,2 and S1. Please modify the manuscript along the lines the reviewers have recommended. As these revisions are quite minor, I expect that you should be able to turn in the revised paper in less than 30 days, if not sooner. If your manuscript was reviewed, you will find the reviewers' comments below.

Laurence Delhaes Raphaël Enaud
When submitting the revised version of your paper, please provide (1) point-by-point responses to the issues raised by the reviewers as file type "Response to Reviewers," not in your cover letter, and (2) a PDF file that indicates the changes from the original submission (by highlighting or underlining the changes) as file type "Marked Up Manuscript -For Review Only". Please use this link to submit your revised manuscript. Detailed instructions on submitting your revised paper are below.

Link Not Available
Thank you for the privilege of reviewing your work. Below you will find instructions from the Microbiology Spectrum editorial office and comments generated during the review.
The ASM Journals program strives for constant improvement in our submission and publication process. Please tell us how we can improve your experience by taking this quick Author Survey. Sincerely,

Silvia Cardona
Editor, Microbiology Spectrum Reviewer comments: Reviewer #2 (Comments for the Author): I thank the authors for considering my comments on the previous version of the manuscript. The manuscript has been improved, however, I still have some concerns. Please see the attached file for my additional comments.
Reviewer #3 (Comments for the Author): Thank you to the authors for addressing my concerns. While not necessary, it might be nice to include a sentence or two in the methods describing the preliminary work done in choosing DNA extraction kits. You did appropriate preliminary work but the current manuscript does not provide the credit for it.

Preparing Revision Guidelines
To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the information as necessary.
Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER.
• Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file.
• Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file. For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process. Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript. " Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me. If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
If your manuscript is accepted for publication, you will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail. Arrangements for payment must be made before your article is published. For a complete list of Publication Fees, including supplemental material costs, please visit our website.

Review for Enaud et al. Lumafactor-Ivacaftor effect on CF lung microbiota-mycobiota and inflammation appears to be linked to Pseudomonas aeruginosa chronic colonization
I thank the authors for considering my comments on the previous version of the manuscript. The manuscript has been improved, however, I still have some concerns. Please see below.

Additional Comments
1) The manuscript still requires additional grammatical review (particularly in the Introduction and Discussion) as there are issues with flow and sentence structure throughout the manuscript which at times makes reading difficult.
2) Line 118 -I recommend changing the sentence that states "restoring a microbial composition closer to that of a healthy individual" to "restoring the pulmonary microbiota to that more closely resembling early disease", given that the use of "healthy" is a bit subjective in this context.
3) Lines 280-282 -The authors state that some genera were "differentially expressed". Please revise this statement to indicate that these were relative abundance measurements rather than measurements of gene expression. Similarly, clarify at the end of the sentence that these measurements were taken at baseline, as the sentence currently suggests that some analysis of change in abundance was performed here.

4)
In my previous review I had commented that the authors should address whether there was a general overall change in the abundance of Gram positive or Gram negative organisms between M0-and M6therapy samples (previous sentence was referred to on Lines 231-241; now Lines 328-334). The authors have provided a table in their response in which they state that 10 bacterial species (4 Gram negative genera and 4 Gram positive genera) had notable differences in relative abundances pre-and duringtherapy. Can the authors please mention this in the text or add a clarifying sentence? 5) Line 298 -The statement that "this finding suggesting a clinical relevance of P. aeruginosa chronic colonization" provides an interpretation of results (e.g. Discussion) rather than a statement of results. Please ensure this is dealt with in the Discussion rather than Results.
6) Tables 1, Table 2, Table S1. In my previous review I commented that Table 1 was initially confusing and required some modification in order to clarify the data presented. The authors have revised this table and created an additional Table S1 which is included in the Supplemental info. The data provided in Table 1 is essentially repeated in both Tables S1 and Table 2 which is not necessary. This information should be stated in a single table to avoid repeating data (also be aware that different numbers, rounded to the nearest whole number vs decimals, are provided for some clinical measurements). This could all be collapsed into Table S1 and Thank you also for the editorial and reviewer comments. We have revised the manuscript to address these comments. The corresponding modifications are visible using the "Track Changes" function in Microsoft Word and we added some comments. We would like to respond to the comments in detail as follows.

Reviewer 2 Comments:
Thank you for taking time to review the article. Please find our point-by-point answers.
1) The manuscript still requires additional grammatical review (particularly in the Introduction and Discussion) as there are issues with flow and sentence structure throughout the manuscript which at times makes reading difficult.
We have submitted our manuscript to a certified English-speaking reviewer. Please find below the certificate of manuscript proofreading.
2) Line 118 -I recommend changing the sentence that states "restoring a microbial composition closer to that of a healthy individual" to "restoring the pulmonary microbiota to that more closely resembling early disease", given that the use of "healthy" is a bit subjective in this context.
Thank you for this suggestion, which we have included in the manuscript.
3) Lines 280-282 -The authors state that some genera were "differentially expressed". Please revise this statement to indicate that these were relative abundance measurements rather than measurements of gene expression. Similarly, clarify at the end of the sentence that these measurements were taken at baseline, as the sentence currently suggests that some analysis of change in abundance was performed here.
We have corrected this point, and rephrased the sentence with : "Among the 16 genera whose relative abundance was significantly different at baseline in patients with FEV1 ≥80%, we observed an overrepresentation of Streptococcus, Porphyromonas, Actinomyces, TM7x and Peptostreptococcus abundances ( Figure 1D)." 4) In my previous review I had commented that the authors should address whether there was a general overall change in the abundance of Gram positive or Gram negative organisms between M0-and M6therapy samples (previous sentence was referred to on Lines 231-241; now Lines 328-334). The authors have provided a table in their response in which they state that 10 bacterial species (4 Gram negative genera and 4 Gram positive genera) had notable differences in relative abundances pre-and duringtherapy. Can the authors please mention this in the text or add a clarifying sentence?
We have included this point by adding the concept of Gram to Table S3 (now Table S2) and modifying the results sentence with: "Comparing M0-M6 microbiota-mycobiota data, DESeq2 analysis revealed a significant increase of Malassezia restricta and a decrease of C. albicans, Capnocytophaga spp., Veillonella spp., TM7x spp., Rothia spp. and Fusobacterium spp. (without a difference evolution between Gram positive and negative organisms) in patients not chronically colonized with P. aeruginosa (Table S3 in the online supplement).
5) Line 298 -The statement that "this finding suggesting a clinical relevance of P. aeruginosa chronic colonization" provides an interpretation of results (e.g. Discussion) rather than a statement of results. Please ensure this is dealt with in the Discussion rather than Results.
We have removed this statement from the results section, as this point is already included in the discussion. Tables 1, Table 2, Table S1. In my previous review I commented that Table 1 was initially confusing and required some modification in order to clarify the data presented . The authors have revised this table  and created an additional Table S1 which is included in the Supplemental info. The data provided in Table 1 is essentially repeated in both Tables S1 and Table 2 which is not necessary. This information should be stated in a single table to avoid repeating data (also be aware that different numbers, rounded to the nearest whole number vs decimals, are provided for some clinical measurements). This could all be collapsed into Table S1 and Table 2 kept in the main body. The authors also need to (i) include units for ppFEV1 and (ii) clarify what is meant by "missing values", as there is no footnote given that explains this.

6)
We combined Table 1 and S1, specified the missing values for each column, and added a footnote to define "missing values". We added the unit for ppFEV1 and checked each number.

Reviewer #3
Thank you for taking time to review the article. Please find our answers.
Thank you to the authors for addressing my concerns. While not necessary, it might be nice to include a sentence or two in the methods describing the preliminary work done in choosing DNA extraction kits. You did appropriate preliminary work but the current manuscript does not provide the credit for it.
We have rephrased the sentence in the methods with : "We have chosen the DNeasy PowerSoil kit (Qiagen, Les Ulis, France) to extract DNA from the samples, after ensuring that it allows the lysis of all bacteria and fungi in our artificial community (see supplementary data). We then followed the manufacturer's protocol, by enhancing the mechanical lysis step with the use of the Precellys evolution (2 cycles of 30 s at 7000 rpm), as previously described".
We hope that this revised version will now be found suitable for publication in Microbiology Spectrum.
Best regards, Thank you for submitting your manuscript to Microbiology Spectrum. As you will see your paper is very close to acceptance. Please modify the manuscript along the lines I have recommended. As these revisions are quite minor, I expect that you should be able to turn in the revised paper in less than 30 days, if not sooner. You will find the reviewers' comments to address below.

Laurence Delhaes Raphaël Enaud
When submitting the revised version of your paper, please provide (1) point-by-point responses to the issues raised by the reviewers as file type "Response to Reviewers," not in your cover letter, and (2) a PDF file that indicates the changes from the original submission (by highlighting or underlining the changes) as file type "Marked Up Manuscript -For Review Only". Please use this link to submit your revised manuscript. Detailed instructions on submitting your revised paper are below.

Link Not Available
Thank you for the privilege of reviewing your work. Below you will find instructions from the Microbiology Spectrum editorial office and comments generated during the review.
The ASM Journals program strives for constant improvement in our submission and publication process. Please tell us how we can improve your experience by taking this quick Author Survey. Sincerely,

Silvia Cardona
Editor, Microbiology Spectrum Pending comments: 1) Please, explain the rationale for the switch between ITS and 18S primers. Please add a sentence to the methods to explain your rationale (you provided it in response to reviewers but not in the paper).
2) Please, explain why the DNA extraction kit was chosen in the methodology section.
3) There are portions of the manuscript that differ between the "marked up" and "clean" versions, particularly in the Discussion section. Please, ensure that the marked-up document and the clean version are the same.

Preparing Revision Guidelines
To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the information as necessary.
Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER. We have revised the manuscript to address editorial comments. The corresponding modifications are visible using the "Track Changes" function in Microsoft Word and we added some comments. We would like to respond to the comments in detail as follows.

Editorial Comments:
1) Please, explain the rationale for the switch between ITS and 18S primers. Please add a sentence to the methods to explain your rationale (you provided it in response to reviewers but not in the paper).
We have revised the relevant part of the methods section to explain our rationale: "qPCR targeting the 16S loci was used to quantify total bacterial loads as previously described (11, 33). Quantification was performed using a standard range of Escherichia coli (ATCC 25922, 2.79 to 2787.1 pg/µL). P. aeruginosa abundance was quantified using a combination of two qPCRs (of oprL and ecfX/gyrB), which have a sensitivity of 100% with a threshold of 10 CFU/mL and a specificity of 100% (39-41).
While the fungal metabarcoding was based on ITS2 amplification, in agreement with published data showing better performance compared to other targets including 18S (42), a published and widely used qPCR based on targeting the fungal 18s region was been chosen to perform fungal loads (43). Quantification was performed using a standard range of Candida albicans DNAs (ATCC 5314, 0.37 pg/µL to 3663.5 pg/µL)."