Dynamic FTIR Spectroscopy for Assessing the Changing Biomolecular Composition of Bacterial Cells During Growth

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In the work, (Manuscript ID: spectroscj-3485626) the FTIR technique was used to probe changes in the biomolecular composition of S. aureus cells in the early- and late-log growth phases. Unfortunately, there is no reference to the latest literature on the discussed topics and no emphasis on the innovativeness of the conducted research, the research is based mainly on the use of one measurement technique. Additionally, several substantive topics require supplementation. 

Detailed comments:

1. In the literature section, please add a review of the latest literature on research on bacteria using the technique used in the experimental section.
2. The introduction must be supplemented with articles on the latest research results on the issues discussed in it (most of the cited works are older than 5 years), only two literature items are from 2023, the rest from later years.

For example, "Analysis of second derivative spectra in FTIR studies of bacteria is becoming standard [23-26]." – please refer to current literature, not e.g. from 2002 or 2013.

3. The data in Table S1 are data that appear in many books, but please move Table S1 to the main manuscript, please update it with the bands indicated in the manuscript – with reference to this work and taking into account the cell growth phases.
4. There is no emphasis on the novelty of the work and the potential application of the research results, which unfortunately are not groundbreaking – please supplement the manuscript and explain the purpose of their publication.

5.       Please standardize the temperature unit to °C (P. 2) or K (P. 3, line 94).

6. Please explain the notation “pca.components_.T function” – provide the algorithms needed for the calculations.
7. Please explain the abbreviation SA6583 (P.4, line 172), and also please check whether it should be SA6583 or SA6538.
8. Figure 1 – please improve the quality, the colors are poorly visible.
9. Which bands appear in the "fingerprint" area and what do they mean?
10. Is it possible to use this method on other strains of bacteria – if so, which bacteria, please support this with literature data.
11. Please label both axes in the Figures presented in the manuscript.

12.    The manuscript should be carefully reread and any editorial mistakes should be corrected (e.g. P.3, line 92 “ddH2O “ – subscript; please standardize the capitalization of letters referring to Figures – e.g. P. 10, lines: 500, 502, 510, 511 – previously capital letters were used; a-helical or α-helical, b-sheet or β-sheet etc.).

After completing the above-mentioned corrections this work will be more readable.   

Author Response

Reviewer #1 states:    

1. In the literature section, please add a review of the latest literature on research on bacteria using the technique used in the experimental section.

We respond: We have added several citations from more recent literature, for example see references 1-5 in the revised manuscript.

Reviewer #1 states:

1. The introduction must be supplemented with articles on the latest research results on the issues discussed in it (most of the cited works are older than 5 years), only two literature items are from 2023, the rest from later years.

For example, "Analysis of second derivative spectra in FTIR studies of bacteria is becoming standard [23-26]." – please refer to current literature, not e.g. from 2002 or 2013.

We respond:

We have removed the older cited papers and replaced with more recent work.

 

Reviewer #1 states:

1. The data in Table S1 are data that appear in many books, but please move Table S1 to the main manuscript, please update it with the bands indicated in the manuscript – with reference to this work and taking into account the cell growth phases.

We respond:

As the reviewer points out, the data in Table S1 appears often in the literature, and even in textbooks. Recent work also places such a Table in the supplementary information, as we have done. We do not agree that moving the table to the main manuscript enhances the manuscript in any way. Especially since we also provide several citations in the main manuscript, and in the Table S1 legend, pointing to assignment of bands in different spectral regions.

We have made considerable reference to the most recent work in this area, and arguably the most comprehensive (reference 30 in revised manuscript). This cited work also cites some of the older literature.

Reviewer #1 states:

There is no emphasis on the novelty of the work and the potential application of the research results, which unfortunately are not groundbreaking – please supplement the manuscript and explain the purpose of their publication.

We respond:

There is considerable text in the manuscript justifying this work. For example, lines 57-65 and lines 66-80. An important result that permeates this paper is that similar work has been undertaken on similar samples to what we used, but different results and conclusions were obtained. The basis for these differences, and what it indicates about the use of the ATR FTIR technique, is an important consideration that more fully highlights the broad applicability of this approach. We believe that this is an important conclusion that is rarely addressed in the literature. Most readers just accept the results of a statistical analysis without appropriate consideration of the raw data used to generate the statistics.  

Reviewer #1 states:  

5. Please standardize the temperature unit to °C (P. 2) or K (P. 3, line 94).

We respond:

We have made the changes standardized to K.

 

Reviewer #1 states:  

6. Please explain the notation “pca.components_.T function” – provide the algorithms needed for the calculations.

We respond:

We modified the text accordingly.

 

Reviewer #1 states:  

7. Please explain the abbreviation SA6583 (P.4, line 172), and also please check whether it should be SA6583 or SA6538.

We respond:

We have corrected the error.

 

Reviewer #1 states:  

8. Figure 1 – please improve the quality, the colors are poorly visible.

We respond:

We are unsure what the reviewer is referring to. The colors seem clearly visible?

 

Reviewer #1 states:  

9. Which bands appear in the "fingerprint" area and what do they mean?

 

We respond:

We assume the reviewer refers to the 1200-700 cm-1 region, or perhaps the 900-600 cm-1 region, or even the 1500-500 cm-1 region? Some of the potential band assignments in this region are indicated in Table S1. The legend in Table S1 also indicates six references, which provide information on the origin of bands in the fingerprint region, below 800 cm-1. In this manuscript bands below 800 cm-1 are not discussed in detail, so the question of relevance arises, as this information is available in a very simple perusal of the cited literature.

 

Reviewer #1 states:  

10. Is it possible to use this method on other strains of bacteria – if so, which bacteria, please support this with literature data.

We respond:

We have added several citations on work undertaken on other strains. See references 1-3.

 

Reviewer #1 states:  

11. Please label both axes in the Figures presented in the manuscript.

We respond:

We feel it is best to describe the contents of the graphs in the figure legends as we have done. Many of the figures contain multiple components (i.e. Absorption spectra, second derivative spectra and differences in second derivative spectra) so labeling the “y-axis” is not possible.

 

Reviewer #1 states:  

12. The manuscript should be carefully reread and any editorial mistakes should be corrected (e.g. P.3, line 92 “ddH2O “ – subscript; please standardize the capitalization of letters referring to Figures – e.g. P. 10, lines: 500, 502, 510, 511 – previously capital letters were used; a-helical or α-helical, b-sheet or β-sheet ).

We respond:

We have made all the necessary corrections.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors conducted an investigation into the biomolecular structure of bacteria during cell growth using FTIR spectroscopy. They presented their findings in detail, and the manuscript is well-organised and easy to understand. Additionally, the references cited in the manuscript are relevant and up to date.

To determine the exact number and position of absorption bands, the authors employed the second derivative method. Have they considered spectral deconvolution, as this would enable semi-quantitative analysis of the spectral bands?

In Figure S1, the decrease in the intensity of the absorption bands in the region of the spectra characteristic of hydroxyl group stretching vibrations with time has been explained as dehydration of the sample. Have the authors considered covering the sample with a glass plate (without applying additional pressure)?

Author Response

Reviewer #2 states: 

The authors conducted an investigation into the biomolecular structure of bacteria during cell growth using FTIR spectroscopy. They presented their findings in detail, and the manuscript is well-organized and easy to understand. Additionally, the references cited in the manuscript are relevant and up to date.

To determine the exact number and position of absorption bands, the authors employed the second derivative method. Have they considered spectral deconvolution, as this would enable semi-quantitative analysis of the spectral bands?

In Figure S1, the decrease in the intensity of the absorption bands in the region of the spectra characteristic of hydroxyl group stretching vibrations with time has been explained as dehydration of the sample. Have the authors considered covering the sample with a glass plate (without applying additional pressure)?

 

We respond:  

We think the reviewer is suggesting keeping the sample in an aqueous environment by covering or sealing? This is a bad idea as the dehydration of the sample is necessary to remove very intense absorption from OH bending vibrations that occur near 1640 cm^-1. If the sample were in solution then a spectrum similar to the green colored one (in Figure S1) would be found. This spectrum is mostly due to water absorption and is of little interest, and obscures features of interest. Measurement of changes in bands during growth, in the 1800-1000 cm-1 region, in the presence of water, would be impossible.

As for the second issue, the reviewer suggests we might apply a Fourier deconvolution rather than use second derivatives. This deconvolution is highly dependent on curve fitting and is often subjective. We feel that such an approach would introduce more problems than it solves and prefer the simpler second derivative approach.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors report on the ATR-FTIR observation of bacterial cells (S. aureus) and analysis of temporal evolution of spectral features.  The manuscript's content matches this journal's scope, and the results are well-presented.  The reviewer would like to recommend it for acceptance after minor revisions.  Each comment is listed below.
(1) The reviewer would like to ask the authors to show the zero point in the 2nd derivative spectra by horizontal dotted (or broken) lines since it would be easier for readers to locate peaks.
(2) The authors clearly show the temporal behaviors of spectral signatures for the secondary structure of proteins.  Is it due to biofilm formation or the metabolism within a bacterial cell itself?  Although it might be difficult due to the absence of spatial resolution, it would be worth mentioning.

Author Response

Reviewer #3 states: 

(1) The reviewer would like to ask the authors to show the zero point in the 2nd derivative spectra by horizontal dotted (or broken) lines since it would be easier for readers to locate peaks.

We respond: 

We have made that change.

 

Reviewer #3 states: 

(2) The authors clearly show the temporal behaviors of spectral signatures for the secondary structure of proteins. Is it due to biofilm formation or the metabolism within a bacterial cell itself? Although it might be difficult due to the absence of spatial resolution, it would be worth mentioning.

 

We respond: 

Temporal changes are not due to biofilm formation. Biofilm formation is secondary to the initial cellular attachment to a surface. Attachment is facilitated by surface proteins and depends on what the substrate is (glass, plastic, cell culture etc.). Cells growing in a shaking flask are not considered "biofilm cells". We added text at line 91 to indicate this.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript has been partially revised.

I have no other comments. However, the introduction has not been significantly updated either. Additionally, the novelty of this work compared to other already published works should be added in the text of the manuscript in the Introduction. Please indicate what techniques were used to study changes occurring during bacterial growth. I disagree with the answer “Most readers just accept the results of a statistical analysis without appropriate consideration of the raw data used to generate the statistics”.

Author Response

The reviewer states:

I have no other comments. However, the introduction has not been significantly updated either.

 

We respond:

We addressed the reviewers concern in the revision, about some reasons for the importance of this manuscript, and pointed to the text on lines 57-80 in the introduction. We also indicate in the first paragraph of the introduction that the work presented in this manuscript is a necessity in any future studies on cells perturbed by drugs or other agents.

 

The reviewer states:

Additionally, the novelty of this work compared to other already published works should be added in the text of the manuscript in the Introduction.

We respond:

We have added text on lines 81-91 to address this concern. We focus on what is arguably the most comprehensive vibrational spectroscopy work that has been undertaken in this area, pointing out some of the deficiencies in that work, and indicate how our manuscript helps address these deficiencies, and why such a description is an important addition to the literature.