A Simple Method for Assessing Diversity and Dynamics of Microbial Community: Comparison of Dairy Phages from Industrial and Spontaneous Fermentation

Round 1

Reviewer 1 Report

The authors of the manuscript entitled "Simple method for assessing diversity and dynamics of microbial community: comparison of dairy phages from industrial and spontaneous fermentation" present their investigation of phage diversity in different curd cheese samples. They found that industrial samples showed a lower diversity compared to in-house specimen. The whole manuscript is rather short and needs some extensions for a better understanding. I would also advise to clearly emphasize the study objective as now it is not really clear what the study motivation was.

Please see below some points that should be addressed before publication.

 

lines 39-45: Disadvantages of NGS are listed. However, no alternatives are presented in detail. The authors should extend this part by listing advantages and disadvantages of their envisaged methodology, otherwise it looks a bit like discrediting NGS.

 

lines 51-54: 25 industrial (all including cow's milk) samples were used and further 14 in-house samples (including cow’s, goat’s or sheep’s milk) were investigated. Please clarify how many of the in-house samples included which of the different milk types as the origin of the milk may have a high impact on the clustering.

 

line 61: please name the primer pairs for better transparency.

 

Figure 1: the non-template control shows an amplicon at low molecular weight as also do most of the other in-house samples. Where does this originate from as the NTC should be completely empty? Please clarify.

 

Figure 5: this figure is really small, please provide a larger image. Additionally, it is not clear what the meaning of the different colors is - please clarify.

 

Supplementary Material: the files attached are not easily accessible, please provide a common file type such as pdf, MS Word, Excel or Powerpoint, or comparable

Author Response

Dear Reviewer,
thank you for your comments concerning our manuscript entitled Simple method for assessing diversity and dynamics of microbial community: comparison of dairy phages from industrial and spontaneous fermentation (applsci-1364728). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our research. We have studied comments carefully and have made corrections. The main corrections in the paper have been included in the manuscript and the responses to the reviewer’s comments are as follows:

Response to Comments from the reviewer:

Point 1: The whole manuscript is rather short and needs some extensions for a better understanding.
Response 1: We have extended the text.

Point 2:

Point 3: Disadvantages of NGS are listed. However, no alternatives are presented in detail. The authors should extend this part by listing advantages and disadvantages of their envisaged methodology, otherwise it looks a bit like discrediting NGS.
Response 3: We did not aim to discredit NGS. We use the technique in our research and value it. However, as we encounter some problems (high costs, a need to fill in the whole throughput) we seek a cheaper alternative or technique that allows preselecting samples prior to NGS.  We changed the text to soften the message.

Point 4: Please clarify how many of the in-house samples included which of the different milk types as the origin of the milk may have a high impact on the clustering.
Response 4: The information was included in the manuscript. We did not found any correlation between milk source and clustering.

Point 5: please name the primer pairs for better transparency.
Response 5: The information was included in the manuscript.

Point 6: Figure 1: the non-template control shows an amplicon at low molecular weight as also do most of the other in-house samples. Where does this originate from as the NTC should be completely empty? Please clarify.
Response 6: The low molecular weight faint bands present also in NTC represent unincorporated primers remaining after reaction. They migrate slower than it is suspected for their length. However, it is typical for ssDNA in comparison with dsDNA [Stellwagen NC, Stellwagen E. Effect of the matrix on DNA electrophoretic mobility. J Chromatogr A. 2009;1216(10):1917-1929. doi:10.1016/j.chroma.2008.11.090].

Point 7: Figure 5: this figure is really small, please provide a larger image. Additionally, it is not clear what the meaning of the different colors is - please clarify.
Response 7: Figure 5 was changed to larger plots. The colors do better visualize overlapping points.

Point 8: Supplementary Material: the files attached are not easily accessible, please provide a common file type such as pdf, MS Word, Excel or Powerpoint, or comparable.
Response 8: Supplementary Material contains a text file with the script for bioinformatic analyses in R environment (applsci.R), a text file with the sequences in FASTA format (File_S1.fas), and data objects for R environment (image_applsci.Rdata)

Reviewer 2 Report

Experiments are from 2017-2018-> How was the situation within the last years?

 

Author Response

Dear Reviewer,
thank you for your comments concerning our manuscript entitled Simple method for assessing diversity and dynamics of microbial community: comparison of dairy phages from industrial and spontaneous fermentation (applsci-1364728).  

Point 1: Experiments are from 2017-2018-> How was the situation within the last years?
Response 1: We have not collected more samples. 

Reviewer 3 Report

In the presente paper, authors compared  the diversity of Lactococcus phages from dairy industrial and spontaneous fermentation processes as different kinds of cheese, using a new method that allowed to study diversity and dynamics of microbial community with well-established and affordable techniques and simple bioinformatics pipeline.

From a scientific point of view, this paper is very interesting because it demonstrates a methodology closer to an industrial routine for screening possible interferents in fermentative processes, and sets precedents for the construction of biosensors or other detection tools.
In my opinion the manuscript is well written. However, the introduction needs to be better grounded in the importance of molecular techniques to better substantiate the importance of more sophisticated techniques in detecting bacteriophage activities that disrupt or reduce the reproducibility of bioprocesses. 
Also, the authors need to revise some English writing errors and improve the written language. An example is line 195, where the word metagemome needs to be replaced by the word metagenome.
With these corrections, I believe the paper can be published in this journal

Author Response

Dear Reviewer,
thank you for your comments concerning our manuscript entitled Simple method for assessing diversity and dynamics of microbial community: comparison of dairy phages from industrial and spontaneous fermentation (applsci-1364728). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our research. We have studied comments carefully and have made corrections. The main corrections in the paper have been included in the manuscript and the responses to the reviewer’s comments are as follows:

Response to Comments from the reviewer:

Point 1: The introduction needs to be better grounded in the importance of molecular techniques to better substantiate the importance of more sophisticated techniques in detecting bacteriophage activities that disrupt or reduce the reproducibility of bioprocesses. 
Response 1: We have extended the introduction.

Point 2: Some English writing errors and improve the written language.
Response 2: The manuscript was checked for writing errors and the language was improved.

Round 2

Reviewer 1 Report

Thank you for revising the manuscript according to the reviewers' comments!

My points were well addressed and the introduction has substantially improved.

I am sorry for being unclear regarding the primers (line 76): I would advise to state the actual primer sequences that were used.