Response of Nicandra physalodes (Linn.) Gaertn. and Its Rhizospheric Organisms to the Selective Pressures of High-Concentration Oxytetracycline, Ciprofloxacin, and Tobramycin
and Chaoxi Chen 1,*
Round 1
Reviewer 1 Report
Growth and biomass accumulation of Nicandra physalodes were evaluated for each antibiotic treatments. Germination rates differed among groups. The height of the plants and enzyme composition of soil responded to antibiotic treatments by inhibition or increase of average height. Similarly earthworm experiments showed variation in the coagulation activities of treatments. The rhizosphere bacterial community differences were determined by 16S V3-V4 region sequencing at different taxonomic levels. The manuscript presents new and interesting data on the effect of antibiotics on soil, plant and microbiome. Minor issues are listed below.
1. Figure 1b and Figure 2: What was the time point for biomass determination?
2. Page 6: Please rephrase this sentence “In this study, the high dosage of three antibiotics may affect the soil enzymes because of the contamination with antibiotics.”
3. Please provide more information about the earthworms used in the experiments like its species name etc.
Author Response
Comments and Suggestions for Authors
- Figure 1b and Figure 2: What was the time point for biomass determination?
Thank you for your constructive suggestions. The time point for biomass determination has been added in the revised manuscript.
- Page 6: Please rephrase this sentence “In this study, the high dosage of three antibiotics may affect the soil enzymes because of the contamination with antibiotics.”
Thank you for your constructive suggestions. The sentence “In this study, the high dosage of three antibiotics may affect the soil enzymes because of the contamination with antibiotics.” has been corrected.
- Please provide more information about the earthworms used in the experiments like its species name etc.
Thank you for your constructive suggestions. The earthworms used in the experiments like their species name were provided in the revised manuscript.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The issue treated in the manuscript is an emerging topic which must be considered by researchers worldwide.
These are my suggestions to improve yor captiving manuscript
1. Please define the species of earthworm (page 2)
2. Please inform the soil umidity and how the water content (and type of water) was controlled.
3. The antibiotics concentration were selected using preset indicators. Please provide the reference even it was yourselves. (written for exemple – “by our group”
4. Page 3 – The period (end of spring and beginning of fall) is suitable for the plant studied? Please write a short phase by saying it.
5. Please italicize the words “in vitro” in all text
6. The use of blood mice needed an committee for use of animals? Please inform the access number or permition
Please answer theses question on discussion:
[1] Why do you study the “raw” drugs and not their metabolites by considering the discharge in the environment is more concentrated with metabolites in place of
[2] considering the drug dissociation in water and the soil pH, how to explain the plant growth as well as in the microbial parameters. I mean if the drug was inactive or need a certain pH to active, do you think it implied in your results?
I let these two questions to contribute to you change terms such as “we speculate that….” To formulate more concise hypothesis.
Author Response
Comments and Suggestions for Authors
- Please define the species of earthworm (page 2)
Thank you for your constructive suggestions. The earthworms used in the experiments like their species name were provided in the revised manuscript.
- Please inform the soil humidity and how the water content (and type of water) was controlled.
Thank you for your constructive suggestions. “After sowing, add the remaining soil evenly and use a shower to moisten the soil with the deionized water, and The whole following experiment was carried out under natural conditions to keep the soil moist.” was provided in the revised manuscript.
- The antibiotic concentrations were selected using preset indicators. Please provide the reference even it was yourselves. (written for exemple – “by our group”
Thank you for your constructive suggestions. It has been corrected in the revised manuscript.
- Page 3 – The period (end of spring and beginning of fall) is suitable for the plant studied? Please write a short phase by saying it.
Thank you for your constructive suggestions.
- Please italicize the words “in vitro” in all text.
Thank you for your constructive suggestions. It has been corrected in the revised manuscript.
- The use of blood mice needed an committee for use of animals? Please inform the access number or permission.
Thank you for your constructive suggestions. The License and Certification Numbers of Laboratory Animals: SCXK(Sichuan)2020-030 and SYXK(Sichuan) 2019-189, The use of blood mice were approved by the Institutional Animal Care and Ethics Committee of Southwest Minzu University.
Please answer these questions on discussion:
[1] Why do you study the “raw” drugs and not their metabolites by considering the discharge in the environment is more concentrated with metabolites in place of
[2] Considering the drug dissociation in water and the soil pH, how to explain the plant growth as well as in the microbial parameters. I mean if the drug was inactive or needed a certain pH to be active, do you think it implied in your results?
Thank you for your constructive suggestions. In the discussion section, we added the sentences as follows: In this study, we just focused on the effect of high-concentration oxytetracycline, ciprofloxacin, and tobramycin on Nicandra physalodes (Linn.) Gaertn. and its rhizospheric organisms, their metabolites and the primary factors such as soil pH and solubility should be elucidated in our ongoing work.
Author Response File: Author Response.pdf
Reviewer 3 Report
The manuscript studies effect of 3 antibiotics on given medicinal plant and its microbiome. It is important and interesting topic.
It has very short introduction highlighting the problem and presenting aim of the study. It could be extended a little more.
In the abstract CK group is mentioned but it is not explained (control?)
When inserting any citations please add space before it. There is many places in the manuscript where spaces are missing.
Material and methods
"and the three antibiotics tested negative by the high performance liquid chromatography..." - what do you mena by 'tested negative'? solvent, flow etc.)
What was the model and manufacturer of HPLC? You should also describe the method of determination (e.g. what kind of detector,
Did you determine soil properties by yourself? If yes, please describe methods and equipment used.
Please use 'kg' instead of 'Kg'
Methods are very weakly described and need strong improvement.
For blood coagulation test earthworms were used and next mouse in mentioned. The procedure mention only material preparation and not the test itself. There is a missing link between earthworm extracts and mouse blood. By the way, do you have approval for mammals usage for the experiment?
Effects of antibiotics on soil enzyme activities - it only states the role of enzymes and the application of manufacturer's protocols. However, more details should be provided and some names of given kits with the their manufacturer.
There is no details on microbial DNA isolation (e.g what kit and methods was used), PCR protocol, purity tests. The specified primers should be followed by the citation of relevant article. There is also a need to explain sequencing (e.g. where it was done, what kind of device was used and its manufacturer).
Which database was used for microbial identification. Is there any outcome of this analysis present in the database (Project number etc.)
For statistics and bioinformatics the name and version of software used need to be provided. There is no information about number of replications and significance levels.
Results could be described more precisely using information about significance of differences (show p-values). The language of statistical description is very inaccurate and unclear.
"PT was significantly decreased (P < 0.01), while TT was not affected in group B compared with the CK group" - when looking of Fig 2. PT levels had increased in comparison CK.
Figures are satisfactory but their font sizes are mostly to small. Please explain the meaning of stars used to indicate statistical differences. Figure captions should provide more details, i.e. explaining each panel of the graph e.g. for Fig.3 a) is APK. b) is UE etc.
Description of Soil enzyme activities is very inaccurate and misleading. Please check carefully your results and re-write this section.
When describing microbial diversity it would be good to provide some percentages for more clear comparison.
Please remember to italize names of genera.
There is hardly any discussion of the results. I also miss convincing conclusions summarizing the results and relating to the aims.
The language could be improved especially when describing results.
Author Response
Comments and Suggestions for Authors
It has a very short introduction highlighting the problem and presenting the aim of the study. It could be extended a little more.
Thank you for your constructive suggestions. We have corrected the problem in the revised manuscript.
In the abstract CK group is mentioned but it is not explained (control?)
Thank you for your constructive suggestions. We have corrected the problem in the revised manuscript.
When inserting any citations please add space before it. There is many places in the manuscript where spaces are missing.
Thank you for your constructive suggestions. We have corrected the problem in the revised manuscript.
"and the three antibiotics tested negative by the high performance liquid chromatography..." - what do you mean by 'tested negative'? solvent, flow etc.)
What was the model and manufacturer of HPLC? You should also describe the method of determination (e.g. what kind of detector,
Did you determine soil properties by yourself? If yes, please describe methods and equipment used.
Thank you for your constructive suggestions. We have corrected the described problem in the revised manuscript.
Please use 'kg' instead of 'Kg'
Thank you for your constructive suggestions. We have corrected the described problem in the revised manuscript.
Methods are very weakly described and need strong improvement.
Thank you for your constructive suggestions. We have corrected the described problem in the revised manuscript.
For blood coagulation test earthworms were used and next mouse in mentioned. The procedure mention only material preparation and not the test itself. There is a missing link between earthworm extracts and mouse blood. By the way, do you have approval for mammals usage for the experiment?
Thank you for your constructive suggestions. We have corrected the described problem in the revised manuscript.
Effects of antibiotics on soil enzyme activities - it only states the role of enzymes and the application of manufacturer's protocols. However, more details should be provided and some names of given kits with the their manufacturer.
Thank you for your constructive suggestions. We have corrected the described problem in the revised manuscript.
There is no details on microbial DNA isolation (e.g what kit and methods was used), PCR protocol, purity tests. The specified primers should be followed by the citation of relevant article. There is also a need to explain sequencing (e.g. where it was done, what kind of device was used and its manufacturer).
Thank you for your constructive suggestions. We have corrected the described problem in the revised manuscript.
Which database was used for microbial identification. Is there any outcome of this analysis present in the database (Project number etc.)
Thank you for your constructive suggestions. We have corrected the described problem in the revised manuscript.
For statistics and bioinformatics the name and version of software used need to be provided. There is no information about number of replications and significance levels.
Thank you for your constructive suggestions. We have corrected the described problem in the revised manuscript.
Results could be described more precisely using information about significance of differences (show p-values). The language of statistical description is very inaccurate and unclear.
"PT was significantly decreased (P < 0.01), while TT was not affected in group B compared with the CK group" - when looking of Fig 2. PT levels had increased in comparison CK.
Thank you for your constructive suggestions. We have corrected the described problem in the revised manuscript.
Figures are satisfactory but their font sizes are mostly to small. Please explain the meaning of stars used to indicate statistical differences. Figure captions should provide more details, i.e. explaining each panel of the graph e.g. for Fig.3 a) is APK. b) is UE etc.
Thank you for your constructive suggestions. We have corrected the described problem in the revised manuscript.
Description of Soil enzyme activities is very inaccurate and misleading. Please check carefully your results and re-write this section.
When describing microbial diversity it would be good to provide some percentages for more clear comparison.
Thank you for your constructive suggestions. We have corrected the described problem in the revised manuscript.
Please remember to italize names of genera.
Thank you for your constructive suggestions. We have corrected the described problem in the revised manuscript.
There is hardly any discussion of the results. I also miss convincing conclusions summarizing the results and relating to the aims.
Thank you for your constructive suggestions. We have corrected the described problem in the revised manuscript.
Author Response File: Author Response.pdf
Round 2
Reviewer 3 Report
Dear Authors,
you have improved your manuscript but there are still issues:
1) statistical analysis - please cite the reference to SPSS (IBM, etc.). Please use upper-case letter for 'p' in p < 0.05 as you use upper-case letter in the results when reporting p-values.
2) You refer to some other works but they are only in Chinese. This makes reader difficult to replicate your study. You should use well accessible references to everybody.
3) You didn't addressed may issues on PCR protocol
4) "Trimmomatic software" has different font size than other text
5) When describing microbial diversity it would be good to provide some percentages (relative abundance) for more clear comparison.
The language is fine.
Author Response
1) statistical analysis - please cite the reference to SPSS (IBM, etc.). Please use upper-case letter for 'p' in p < 0.05 as you use upper-case letter in the results when reporting p-values.
Thank you for your suggestion! “(IBM, Version 19.0)” and “The values of P< 0.05 and P< 0.01 are considered to be statistically significant and extremely distinct differences, respectively.” is modified in “Statistical analysis”.
2) You refer to some other works but they are only in Chinese. This makes reader difficult to replicate your study. You should use well accessible references to everybody.
Thank you for your suggestion! New references have replaced the former ones.
3) You didn't address may issues on PCR protocol
Thank you for your suggestion! Issues on PCR protocol have been added.
4) "Trimmomatic software" has different font size than other text.
The font size of “Trimmomatic software”in “Effects of antibiotics on Nicandra physalodes (Linn.) Gaertn. rhizosphere microorganism diversity” has been changed.
5) When describing microbial diversity it would be good to provide some percentages (relative abundance) for more clear comparison.
Thank you for your suggestion! some percentages of relative abundance have been added.
