Optimizing synthetic cystic fibrosis sputum media for growth of non-typeable Haemophilus influenzae
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Non-typeable Haemophilus influenzae (NTHi) is an early pathogen isolated from the lungs of children with cystic fibrosis (CF). However, its role in the progression of CF lung infection is poorly understood. Additionally, whether it forms biofilms in the lungs of people with CF is an open question. The development of synthetic CF sputum media (SCFM) has given key insights into the microbiology of later CF pathogens, Pseudomonas aeruginosa and Staphylococcus aureus , through replicating the chemical composition of CF sputum. However, the growth of NTHi in these media has not previously been reported. We show that NTHi grows poorly in three variants of SCFM commonly used to induce in vivo -like growth of P. aeruginosa and S. aureus (SCFM1, SCFM2 and SCFM3). The addition of NAD and haemin to SCFM1 and SCFM2 promoted the planktonic growth and biofilm formation of both laboratory and clinical NTHi isolates, and we were able to develop a modified variant of SCFM2 that allows culture of NTHis. We show that NTHi cannot be identified in an established ex vivo model of CF infection, which uses SCFM and porcine bronchiolar tissue. This may in part be due to the presence of endogenous bacteria on the pig lung tissue, which outcompete NTHi, but the lack of selective agar to isolate NTHi from endogenous bacteria, and the fact that NTHi is an exclusively human pathogen, makes it hard to conclude that this is the case. Through spiking modified SCFM2 with filter-sterilized lung homogenate, biofilm growth of clinical NTHi isolates was enhanced. Our results highlight that there are crucial components present in the lung tissue, which NTHi require for growth, which are not present in any published variant of SCFM from the Palmer et al. Endres and Konstan in JAMA (2022;137:191-1) lineage. Our results may inform future modifications to SCFM recipes to truly mimic the environment of CF lung sputum and thus, to facilitate the study of a wide range of CF pathogens.
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I am pleased to tell you that your article has now been accepted for publication in Access Microbiology. This is a study that would be of interest to the field and community.
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Thank you very much for submitting your manuscript to Access Microbiology. It has now been reviewed by two experts in the field, whose comments are attached at the bottom of this email. In general, they both agree that this is a valuable research contribution, however they have pointed out some concerns that need to be clarified and suggestions for improvement of the manuscript. Please address them in a revised manuscript. Please provide a revised version of the manuscript (including a tracked-changes document) along with a point-by-point response to the reviewer comments within 2 months.
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Comments to Author
1. Methodological rigour, reproducibility and availability of underlying data Line 121, Clarify why the phenol extraction of DNA was carried out. 2. Presentation of results Results were presented clearly with easy to understand figures. It would be beneficial to clarify what the different shapes of data points mean in Figures 1,2, 4, 5, 7, and Supplementary Figure 3. Please provide clarity in the sentence beginning "NTHi binds host mucin via sialic acid..." on line 218. Why doesSCFM2 increase carrying capacity for NCTC 11315 but decrease it for NCTC 12975? Line 299 Line 306, While the comparison to P. aeruginosa is interesting, it would help to clarify whether similar eDNA sensitivity has been observed in NTHi biofilms or if this is purely speculative. Line 315, The hypothesis could be stated …
Comments to Author
1. Methodological rigour, reproducibility and availability of underlying data Line 121, Clarify why the phenol extraction of DNA was carried out. 2. Presentation of results Results were presented clearly with easy to understand figures. It would be beneficial to clarify what the different shapes of data points mean in Figures 1,2, 4, 5, 7, and Supplementary Figure 3. Please provide clarity in the sentence beginning "NTHi binds host mucin via sialic acid..." on line 218. Why doesSCFM2 increase carrying capacity for NCTC 11315 but decrease it for NCTC 12975? Line 299 Line 306, While the comparison to P. aeruginosa is interesting, it would help to clarify whether similar eDNA sensitivity has been observed in NTHi biofilms or if this is purely speculative. Line 315, The hypothesis could be stated more directly — perhaps rephrased as: 'We hypothesized that SCFM2.SA.XV, designed to mimic the CF lung environment, would support the growth of clinical NTHi isolates. Line 318, Consider trimming repetitive phrasing of the word 'isolates' and breaking up longer sentences for improved readability Line 334, Consider clarifying whether there are any differences in commensals between human lung and pig lung microbiomes, which could have an effect on the growth and behaviour of CF pathogens such as NTHi. Line 337, Clarify whether autofluorescence of the lung tissue was taken into consideration when measuring fluorescence of GFP-tagged strains. 3. How the style and organization of the paper communicates and represents key findings The layout of the paper is clear to follow and understand, with major findings being communicated effectively. The introduction explains the relevance of NTHi in Cystic fibrosis and provides a strong rationale for the study 4. Literature analysis or discussion The discussion is clearly set out and summarises results from the paper in a way that is easy to follow. Line 403, it is mentioned that other sources of eDNA should be explored, however it would be useful to clarify what alternative sources will be used. 5. Any other relevant comments The paper is a very interesting read and clearly explains methods and results well.
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Very good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
Authors state that they ''show NTHi grows poorly'' in the three SCFM variants SCFM1, SCFM2 and SCFM3 (Line 15-17). However, NTHi growth in SCFM3 is not compared to the growth in SCFM1 and SCFM2 in the paper. To demonstrate that, all three SCFM media should be tested at the same time and data presented on the same graph. SCFM3 contains NAD+, needed for NTHi growth, so what was authors' rationale to excluded it from the majority of the experiments present in the paper? According to Fig.4, NTHi growth profiles in SCFM3 are pretty similar to those in the modified SCFM2.SA.XV. Hence, more explanation or rewording of the statement on Line 303 is needed. Have the authors considered modifying SCFM3 by adding hemin to it? Line 122-124 The authors used UV light to sterilise the salmon sperm DNA to decrease …
Comments to Author
Authors state that they ''show NTHi grows poorly'' in the three SCFM variants SCFM1, SCFM2 and SCFM3 (Line 15-17). However, NTHi growth in SCFM3 is not compared to the growth in SCFM1 and SCFM2 in the paper. To demonstrate that, all three SCFM media should be tested at the same time and data presented on the same graph. SCFM3 contains NAD+, needed for NTHi growth, so what was authors' rationale to excluded it from the majority of the experiments present in the paper? According to Fig.4, NTHi growth profiles in SCFM3 are pretty similar to those in the modified SCFM2.SA.XV. Hence, more explanation or rewording of the statement on Line 303 is needed. Have the authors considered modifying SCFM3 by adding hemin to it? Line 122-124 The authors used UV light to sterilise the salmon sperm DNA to decrease hazard from phenol extraction and shorten the time for medium preparation. However, UV irradiation of DNA not only causes DNA damages, but can also lead to the production of free radicals, which can impact bacterial growth and biofilm formation. Have the authors considered this as a factor affecting their results and have they assessed the effect of the UV sterilisation on the eDNA? Fig.5 - NTHi growth in SCFM2.SA.XV is compared only to SCFM1. In addition, the authors have to compare growth in SCFM2.SA.XV to the control SCFM2 and SCFM2.SA media, as well as to SCFM3. Line 158-160 Could the authors explain their rationale behind scraping the base of the biofilms wells during the PBS washing steps, the subsequent sonication of the biofilms and the two separate OD600 measurements during the CV biofilm assay? It's not clear if the crystal violet staining was done after the sonication of the biofilms. If so, the assay needs to be repeated, as scraping the bottom of the biofilm wells and/or sonication would disrupt the biofilm formed there, increasing the chance of washing it off the wells during the washing steps and resulting in incorrect biofilm measurement. The authors state that ''SCFM2.SA.XV was the only media variant to significantly promote biofilm formation, although in only one laboratory strain'' (line 258), which according to Fig.2 is NCTC 12699. However, after presenting the ECM assay results, the authors state that ''biofilm formation in SCFM2.SA.XV was most apparent with NCTC 4560 and NTCT 11315'' (line 269-270). Also, in Line 274-275 the authors suggest that NCTC 12699 could be a ''weak biofilm former in SCFM'' based on Fig.3 data, but this contradicts the results shown in Fig.2. Could the authors comment on why the ECM data does not reflect the CV biofilm data (especially about the two strains showing high ECM levels but low biofilms from the CV assay)? The authors mention NTHi as an early CF pathogen found in samples from children with CF (line 11, 43-45, 382-383). A useful point of discussion would be on the potential differences between the CF lung conditions early and later in life and whether this could affect NTHi growth in SCFM, as the SCFM composition is based on sputum samples from adults (PMID: 17873029). Line 32 The full name of CFTR is not written right after the abbreviation. Line 51-54 These last sentences of the second paragraph of the introduction can be moved to the last paragraph of the introduction, so the story flows from introducing Hi as a CF pathogen to explaining what Hi is. Line 79 Remove ''of'' from ''to gain an understanding of if H. influenzae form biofilms''. Line 2010-2020 give the reasoning for the improvements of the SCFM, so it could come first in the paragraph, followed by line 206-209 which briefly explains what was done and then the paragraph starting from line 221 presenting the results. Line 217-2019 The order of the sentences needs to be swapped to improve the flow of the story. Line 229 Replace ''in particularly'' with ''in particular''. Line 252 The word ''seen'' is accidentally repeated twice in ''was seen only seen in NTCC 12699''. Line 267 Reference to Fig.3 Line 272 Replace ''with'' with ''of'' in ''wile fluorescence with NCTC 12699''. Line 274 ''This suggest NCTC 12699 and NCTC 11315 do not produce as much extracellular matrix'' - did the authors mean NCTC 12975 instead of NCTC 11315? Line 299 Refer to Fig.4 Line 318 The word ''isolates'' is repeated twice in ''three isolates (ID8, ID21 and ID37) isolates''. Line 337 Add ''of'' to ''a lack selective media''. Line 354 Replace ''maybe'' with ''may'' in ''but maybe also be''. Line 367 Strain ID37, which growth is increased too (Fig.7), is not mentioned. Line 386 Add the name of the modified medium in brackets. Line 397 Remove the second ''the'' from ''for the enhanced the growth''.
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Very good
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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