Detection of Microbial Nitroreductase Activity by Monitoring Exogenous Volatile Organic Compound Production Using HS-SPME-GC-MS
Round 1
Reviewer 1 Report
Abstract:
Guidelines: Abstract can have 200 words according to guidelines and should contain background, methods, results and conclusion. --> More Details should be added
Introduction
Guidelines: The current state of the research field should be reviewed carefully and key publications cited. Please highlight controversial and diverging hypotheses when necessary. --> There is no classification from the literature of what exactly was done and why this paper closes this gap
Guidelines: Finally, briefly mention the main aim of the work and highlight the main conclusions. Keep the introduction comprehensible to scientists working outside the topic of the paper -->Conclusion is missing
In this technique, enzymatic hydrolysis of an appropriate substrate by a microorganism generates a volatile organic compound (VOC) which is subsequently absorbed onto a SPME fibre located within the headspace of a sample vial containing the microorganism of interest in a suitable growth medium. 28 The VOC is then thermally desorbed from the SPME fibre and analysed by GC-MS. --> this is more a part of methods than of introduction
line 33 anilines: which ones are of interest? Are there only these sources (8-14) on this topic? What are the volatile markers that were found there? This would explain why this paper investigates this and what is different about it compared to the already published papers.
in [8] the authors have determined 3-fluoroanilines. Why do the authors select the mentioned two analytes?
line 39: Is there a study that the selected analytes can not be found in "healthy" blood?
line48: It would make sense to write "pathogenic" in the abstract and even title
line 49 and 50: why are the markes of interest? is there any literature to substantiate this selection?
Materials and reagents:
Guidelines Materials and Methods: They should be described with sufficient detail to allow others to replicate and build on published results. New methods and protocols should be described in detail while well-established methods can be briefly described and appropriately cited. Give the name and version of any software used and make clear whether computer code used is available.--> the text should be adapted
line 56 ff: instead of order numbers the CAS numberes, the degree of purity and if necessary the manufacturer should be used. minimum one order number is wrong
line 60: Merck Millipore, XY, Germany
Instrumentation:
Was also the same method of [7] used in this work?
Results and Discussion:
line 90: please go mor into detail: was the double determination independent or 2x one sample measured? was it completely independent incl. preparation or how were the samples separated?
Table 1: units are missing for retention time Quantitative m/z, LOD, LOQ linear fucntion
line 93: What do you mean with "identical positive responses"? Table 2 ist not mentioned in the text of this paragraph
line 110ff:
Can you discuss the results more detailed? Why do you thin the results are as shown? Is there any evidence in the literature that these tendencies exist?
Sometimes the fluctuations in the classes are very large. Does that have a reason?
Was it tested whether the samples really grew well? Double determination with 2 samples instead of 2x from one sample with biological material would be better because biological variances can be represented in this way.
line 126: Which VOC and how much?
line 182: Table 3. is not mentioned in the text
Generall: Supplementary material is not described in the text
Author Response
Reviewer 1:
Abstract:
Guidelines: Abstract can have 200 words according to guidelines and should contain background, methods, results and conclusion. --> More Details should be added
Author response: the abstract has been extensively re-worked as per reviewer suggestion.
Introduction
Guidelines: The current state of the research field should be reviewed carefully and key publications cited. Please highlight controversial and diverging hypotheses when necessary. --> There is no classification from the literature of what exactly was done and why this paper closes this gap
Author response: The introduction carefully highlights the main uses of enzyme substrates to liberate VOCs. Some additional text has been added that broadens the purpose of the paper.
Guidelines: Finally, briefly mention the main aim of the work and highlight the main conclusions. Keep the introduction comprehensible to scientists working outside the topic of the paper -->Conclusion is missing
Author response: The paper has a conclusion. The introduction has been extended to provide more understanding for scientists outside the field.
In this technique, enzymatic hydrolysis of an appropriate substrate by a microorganism generates a volatile organic compound (VOC) which is subsequently absorbed onto a SPME fibre located within the headspace of a sample vial containing the microorganism of interest in a suitable growth medium. 28 The VOC is then thermally desorbed from the SPME fibre and analysed by GC-MS. --> this is more a part of methods than of introduction
Author response: This section has been moved to Methods/Instrumentation.
line 33 anilines: which ones are of interest? Are there only these sources (8-14) on this topic? What are the volatile markers that were found there? This would explain why this paper investigates this and what is different about it compared to the already published papers.
Author response: Text has been added. The main approach for detecting enzyme activity in bacteria is by using enzyme substrates that liberate a chromophore or fluorophore. The generation of the colour or fluorescence can take longer than what we have determined in the paper; typically 24 hours.
in [8] the authors have determined 3-fluoroanilines. Why do the authors select the mentioned two analytes?
Author response: Our aim is to generate VOCs that are unlikely to naturally occur, thereby avoiding false positives.
line 39: Is there a study that the selected analytes can not be found in "healthy" blood?
Author response: not that we aware, but by using enzyme substrates that liberate VOCs with some functionality e.g. an amino//fluorine combination would in high probability never occur naturally. However, we also did extensive blank and cross-checking to ensure that no VOCs were generated naturally, with the same functionality.
line48: It would make sense to write "pathogenic" in the abstract and even title
Author response: added in abstract
line 49 and 50: why are the markes of interest? is there any literature to substantiate this selection?
Author response: Our reason for selecting these VOCs / substrates was based on cost; they are all readily available at low cost. So any developed universal microbial detection system would likely be more adopted if the cost of the reagents is low.
Materials and reagents:
Guidelines Materials and Methods: They should be described with sufficient detail to allow others to replicate and build on published results. New methods and protocols should be described in detail while well-established methods can be briefly described and appropriately cited. Give the name and version of any software used and make clear whether computer code used is available.--> the text should be adapted
Author response: The text has been amended and details of the instrumentation and operation of the SPME added.
line 56 ff: instead of order numbers the CAS numberes, the degree of purity and if necessary the manufacturer should be used. minimum one order number is wrong
Author response: these have all been amended.
line 60: Merck Millipore, XY, Germany
Author response: details added
Instrumentation:
Was also the same method of [7] used in this work?
Author response: the exact version for this research has been added
Results and Discussion:
line 90: please go mor into detail: was the double determination independent or 2x one sample measured? was it completely independent incl. preparation or how were the samples separated?
Author response: 2 from the same vial; that is why most of the VOCs determined are precise.
Table 1: units are missing for retention time Quantitative m/z, LOD, LOQ linear function
Author response: all now added
line 93: What do you mean with "identical positive responses"? Table 2 ist not mentioned in the text of this paragraph
Author response: precise is a better word; Table 2 added in text
line 110ff:
Can you discuss the results more detailed? Why do you thin the results are as shown? Is there any evidence in the literature that these tendencies exist?
Author response: We are not aware of any similar study, so difficult to compare.
Sometimes the fluctuations in the classes are very large. Does that have a reason?
Author response: I would disagree, these data are all precise. As the bacteria are all alive and producing VOCs, based on excess substrate, the duplicate values are good (in my opinion).
Was it tested whether the samples really grew well? Double determination with 2 samples instead of 2x from one sample with biological material would be better because biological variances can be represented in this way.
Author response: samples all grew. Previous work would have indicated less precise VOC data for independent samples. The good precision obtained here is because the same vial has been sampled.
line 126: Which VOC and how much?
Author response: 2-fluoroaniline; the concentration detected was not a fixed ratio compared to aniline.
line 182: Table 3. is not mentioned in the text
Author response: it was already mentioned, just before Figure 1 in the text.
Generall: Supplementary material is not described in the text
Author response: I have now removed the Supplementary Material all together and added to the text where it will be more valuable to view each Figure alongside the others.
Reviewer 2 Report
The manuscript deals with a very interesting and topical issue in contemporary food and clinical science: detection of microbial nitroreductase activity by monitoring exogenous volatile organic compound production using HS-SPME-GC-MS. Unfortunately, the important matter of the study suffers by the poor preparation of the very manuscript. I have some general notes and recommendations:
- In “Abstract”: the authors should add 1-2 sentences at the end of this section on the applicability of the obtained research results as well as on the future research perspectives.
- The manuscript needs a consultation with a native English speaker to improve its grammar and style.
- In “Materials and methods”: the authors should explain the selection of test pathogenic microorganisms as well as why some genera have less representatives in the examination than others.
- In “Results and discussion”: there is no discussion part.
- In “Results and discussion”: the authors should have included more representatives from one and the same genus in order to make general conclusions on the existence of intra genus concurrence; such conclusions cannot be drawn from testing only two representatives of a genus.
- In “Conclusion”: the authors should add 1-2 sentences at the end of this section on the applicability of the obtained research results as well as on the future research perspectives.
Author Response
Reviewer 2
The manuscript deals with a very interesting and topical issue in contemporary food and clinical science: detection of microbial nitroreductase activity by monitoring exogenous volatile organic compound production using HS-SPME-GC-MS. Unfortunately, the important matter of the study suffers by the poor preparation of the very manuscript. I have some general notes and recommendations:
- In “Abstract”: the authors should add 1-2 sentences at the end of this section on the applicability of the obtained research results as well as on the future research perspectives.
Author response: The abstract has been amended
- The manuscript needs a consultation with a native English speaker to improve its grammar and style.
Author response: ok, we have done this
- In “Materials and methods”: the authors should explain the selection of test pathogenic microorganisms as well as why some genera have less representatives in the examination than others.
Author response: The test pathogenic bacteria were selected on the basis of their importance in a clinically relevant setting ie an NHS Hospital.
- In “Results and discussion”: there is no discussion part.
Author response: There is not a specific section on discussion; the entire section is written as results and discussion.
- In “Results and discussion”: the authors should have included more representatives from one and the same genus in order to make general conclusions on the existence of intra genus concurrence; such conclusions cannot be drawn from testing only two representatives of a genus.
Author response: The examples selected were chosen by an NHS medical professional in microbiology.
- In “Conclusion”: the authors should add 1-2 sentences at the end of this section on the applicability of the obtained research results as well as on the future research perspectives.
Author response: additional text has been added, with some re-writing as well.
Round 2
Reviewer 2 Report
The authors have addressed some of my previous notes and recommendations. Unfortunately, they did not address all of them, thus, I do not find the manuscript to be significantly improved. I cannot but point out the following flaws of the manuscript:
- In “Materials and methods”: the authors should explain and include the explanation in the very text of the manuscript the selection of test pathogenic microorganisms as well as why some genera have less representatives in the examination than others.
- In “Results and discussion”: there is no discussion part. I know that the name of the section is “Results and discussion”, but the Discussion element implies to include comparison of the obtained results with research results of other research teams having performed similar or connected experiments and to draw some significant conclusions out of the comparison made. The present manuscript lacks that.
- In “Results and discussion”: the authors should have included more representatives from one and the same genus in order to make general conclusions on the existence of intra genus concurrence; such conclusions cannot be drawn from testing only two representatives of a genus.
- The aim of the manuscript is poorly formulated and structured. I suggest putting the previous results obtained and the explanations needed right before formulating the aim of the manuscript in one single sentence.
- The numbering of the sections and subsections in the manuscript needs correction.
Author Response
The authors have addressed some of my previous notes and recommendations. Unfortunately, they did not address all of them, thus, I do not find the manuscript to be significantly improved. I cannot but point out the following flaws of the manuscript:
- In “Materials and methods”: the authors should explain and include the explanation in the very text of the manuscript the selection of test pathogenic microorganisms as well as why some genera have less representatives in the examination than others.
Response: Text has been added to the methods section to state that : “Microorganisms were selected to represent a wide range of pathogens responsible for a variety of infections including blood stream infections and gastroenteritis. For the most common pathogenic species encountered in the clinical microbiology laboratory (such as Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa), more than one example of each was included.
- In “Results and discussion”: there is no discussion part. I know that the name of the section is “Results and discussion”, but the Discussion element implies to include comparison of the obtained results with research results of other research teams having performed similar or connected experiments and to draw some significant conclusions out of the comparison made. The present manuscript lacks that.
Response: We have added text (and 5 new references) at the end of this section to discuss related studies in some detail to show what has been achieved in this field.
- In “Results and discussion”: the authors should have included more representatives from one and the same genus in order to make general conclusions on the existence of intra genus concurrence; such conclusions cannot be drawn from testing only two representatives of a genus.
Response: We generally considered it more useful to have a wide range of species rather than having several representatives of each individual species. As we were seeking universal detection of a very wide range of species, we had no ambition to achieve species differentiation and therefore no particular interest in achieving concurrence among members of a particular species or genus. If this was our aim, we agree that inclusion of multiple representatives would have been essential. It may be relevant to note that in several previously published studies (now quoted in the discussion) far fewer species have been tested – with usually only one representative of each species.
- The aim of the manuscript is poorly formulated and structured. I suggest putting the previous results obtained and the explanations needed right before formulating the aim of the manuscript in one single sentence.
Response: We agree this is a fair point. We have a clear and concise aim at the end of the introduction.
- The numbering of the sections and subsections in the manuscript needs correction.
Response, now amended
Round 3
Reviewer 2 Report
This time the authors have taken into consideration my good-intended notes and recommendations and have significantly improved the manuscript. I have no further notes and recommendations on it.
