Human Milk Oligosaccharide-Stimulated Bifidobacterium Species Contribute to Prevent Later Respiratory Tract Infections
Round 1
Reviewer 1 Report
The study concerns the use of human milk oligosaccharide in stimulating Bifidobacteria in the early stages of the child's life and helping to prevent respiratory tract infections.
The study is interesting and well presented. However, sampling is limited and the study could represent a preliminary analysis.
Minor comments:
- Introduce a section of abbreviations.
- Line 49, do not use the undefined abbreviation.
- I suggest moving Figure 1 of the Flow chart near lines 120-130.
- Line 486, "with ..." in uppercase.
Author Response
We thank the reviewer for the feedback and constructive suggestions.
The study is interesting and well presented. However, sampling is limited and the study could represent a preliminary analysis.
Response: We appreciate this comment and do mention the limited sample size as part of the study limitations.
Minor comments:
- Introduce a section of abbreviations.
Response: We now included a list of abbreviations at the end of the manuscript, from line 517.
- Line 49, do not use the undefined abbreviation.
Response: We now included in line 44 the abbreviation.
- I suggest moving Figure 1 of the Flow chart near lines 120-130.
Response: We moved Figure 1 as suggested.
- Line 486, "with ..." in uppercase.
Response: We corrected this and an additional typo.
Reviewer 2 Report
Minor remarks
Line 22 please explain “2-HMO” at the first use
General comments to the section: In vitro fermentation assay and analysis
Conditions applied for routine cultivation of HT-29 and Caco-2 should be given, the number of cells loaded into wells should be expressed as “cells per well”. Number of Salmonella cells applied on enterocytes should be given in “cells per well” or “bacterial cells per one epithelial cell”. The applied by Authors “cells per millilitre” is confusing since the working volume in a multi-well plate can differ depending on an experimental design.
What was the experimental control for Salmonella invasion assay? I mean the control showing that Salmonella cells do invade epithelial cells. Did Authors confirm the efficacy of gentamycin treatment?
Line 163 please specify what is MRS API medium? Please provide composition and/or manufacturer if it differs form standard MRS medium
Line 164 Please indicate how anaerobic conditions were obtained
Line 166-167 “An OD of 0.1 of each preparation was added to 10 ml of DMEM cell culture media” – I don’t understand. Do you mean that DMEM medium inoculated with B. infantis had OD600 = 0.1 compared to non-inoculated medium? Please reformulate the sentence.
Line 183-187 – please provide volume of media loaded per well, and a number of Salmonella cells per well.
Line 187-188 please describe how results of NFkB stimulation were calculated and expressed.
Line 191 clearly state what medium Salmonella was grown in
Line 195 please give the number of Salmonella cells per well
Line 199-200 “Caco-2 cells were lysed with water” what water?
Line 409 please use either “bifidobacteria” (a regular font) or “Bifidobacterium” (italics)
Line 419 Authors did not investigated the attachment of Salmonella cells to Caco-2 (by the way, it would be interesting to compare the ratio of the adhered cells to the invading ones). Please correct the sentence.
Line 486 “With such limitations”
Figure 6d – please change the y axis title as it is misleading
Author Response
We thank the reviewer for constructive feedback and suggestions.
here our point by point responses in blue.
Minor remarks
Line 22 please explain “2-HMO” at the first use
Response: We adjusted the text accordingly.
General comments to the section: In vitro fermentation assay and analysis
Conditions applied for routine cultivation of HT-29 and Caco-2 should be given, the number of cells loaded into wells should be expressed as “cells per well”. Number of Salmonella cells applied on enterocytes should be given in “cells per well” or “bacterial cells per one epithelial cell”. The applied by Authors “cells per millilitre” is confusing since the working volume in a multi-well plate can differ depending on an experimental design.
Response: We adjusted the experimental descriptions and provide now the ‘cells per well’ information.
What was the experimental control for Salmonella invasion assay? I mean the control showing that Salmonella cells do invade epithelial cells. Did Authors confirm the efficacy of gentamycin treatment?
Response: The efficacy of gentamycin at 100 microgram/ml was tested during the setting up of the protocol.
Line 163 please specify what is MRS API medium? Please provide composition and/or manufacturer if it differs form standard MRS medium
Response: We now clarified that MRS was a glucose free medium otherwise like standard MRS medium.
Line 164 Please indicate how anaerobic conditions were obtained
Response: We included now how the anaerobic conditions were established.
Line 166-167 “An OD of 0.1 of each preparation was added to 10 ml of DMEM cell culture media” – I don’t understand. Do you mean that DMEM medium inoculated with B. infantis had OD600 = 0.1 compared to non-inoculated medium? Please reformulate the sentence.
Response: We rephrased the sentence to clarify that a final OD of 0.1 was achieved in DMEM culture media.
Line 183-187 – please provide volume of media loaded per well, and a number of Salmonella cells per well.
Response: We changed the dimensions to cells per well.
Line 187-188 please describe how results of NFkB stimulation were calculated and expressed.
Response: We specified now that NFkB reporter luminescence was measured according to instructions provided with the measurement kit and that we expressed the data relative to the glucose control.
Line 191 clearly state what medium Salmonella was grown in
Response: We added the missing information on the growth medium.
Line 195 please give the number of Salmonella cells per well
Response: We expressed now in cells per well.
Line 199-200 “Caco-2 cells were lysed with water” what water?
Response: We now specific ‘sterile MilliQ-water’.
Line 409 please use either “bifidobacteria” (a regular font) or “Bifidobacterium” (italics)
Response: We corrected this typo.
Line 419 Authors did not investigated the attachment of Salmonella cells to Caco-2 (by the way, it would be interesting to compare the ratio of the adhered cells to the invading ones). Please correct the sentence.
Response: We now corrected the text.
Line 486 “With such limitations”
Response: We corrected these typos.
Figure 6d – please change the y axis title as it is misleading
Response: We changed the legend to “% of Glc” to be consistent and clear.
