In Vitro Antibacterial Activity of Selected South African Plants against Drug-Resistant Staphylococci Isolated from Clinical Cases of Bovine Mastitis
Round 1
Reviewer 1 Report
The study evaluated 'In vitro antibacterial activity of selected nine plants against bovine mastitis'.
This original manuscript was prepared well with all parts of the manuscript and discussed with the results of the study very deeply and detail. But it needs some minor corrections, please see within the pdf file.
Comments for author File: 
Comments.pdf
Author Response
Thank you to the reviewer for the constructive comments provided, Responses to each query have been provided in the attached pdf file.
Author Response File: Author Response.pdf
Reviewer 2 Report
Overall Manuscript is moderate and can be publish with minor corrections. Few of the results are not supported with proper results.
Line 203: Reference is incomplete no page no, neither article ID, pls incorporate
Line No 106: What is the selection criteria for the plants as they belongs to different families. Is there any ethnomedicinal background?
Line 202: Why sterile distilled water served as sterile control when author is using acetone and alcohol as solvent for extraction? Not applicable
Line: 318: Acetone extract is showing the compounds but ethanol extract is not showing. May be the solvent system is not correct for ethanol extract as all the solute is near start line. Nt clear tlc report. Therefore, difficult to interpretate the results of tlc and bioefficacies. The Figure 1 and other tlc results are not proper ligand supported. It is difficult to understand what author want to explain.
Line 276: BEA is abbreviated and complete solvent system is not also reported in material methods. Pls incorporate. All the triple results tables as annexures must be submitted.
Antibacterial activity is also not supported with photographs so not able to understand the results. Please support the data with photo plates with zone of inhibitions
Moderate English can be improved.
Author Response
Overall Manuscript is moderate and can be published with minor corrections. Few of the results are not supported with proper results.
Response: Thank you very much for your observations and comments. The results have been improved.
Line 203: Reference is incomplete no page no, neither article ID, pls incorporate
Response: The reference has been completed with inclusion of article ID.
Line No 106: What are the selection criteria for the plants as they belong to different families? Is there any ethnomedicinal background?
Response: The selection criteria have been included in section 2.1.
Line 202: Why sterile distilled water served as sterile control when author is using acetone and alcohol as solvent for extraction? Not applicable
Response: This has been excluded.
Line: 318: Acetone extract is showing the compounds but ethanol extract is not showing. May be the solvent system is not correct for ethanol extract as all the solute is near start line. Nt clear tlc report. Therefore, difficult to interpret the results of tlc and bioefficacies. The Figure 1 and other tlc results are not proper ligand supported. It is difficult to understand what author want to explain.
Response: Thank you for pointing this out; the figures have been improved.
Line 276: BEA is abbreviated and complete solvent system is not also reported in material methods. Pls incorporate. All the triple results tables as annexures must be submitted.
Antibacterial activity is also not supported with photographs so not able to understand the results. Response: BEA was first mentioned in section 2.5 and was referred to in section 2.7.1. The whole solvent system was described in section 2.5 and we judged it appropriate to only refer to it in section 2.7.1. which described the materials and methods. The tables have been submitted as annexures with this response, and any further raw data required by readers is available from the authors on request.
Please support the data with photo plates with zone of inhibitions
Response: The available photos were added.
Reviewer 3 Report
Your paper, with the title -In vitro antibacterial activity of selected South African plants against drug-resistant staphylococci isolated from clinical cases of bovine mastitis- has a few interesting experimental observations developed in the laboratory by different methods.
To improve the quality of the manuscript I will suggest some modifies:
-In the introduction chapter, please more references about the cytotoxicity of the plant extracts to be in correlation with the manuscript objectives.
-Linea 46-47 –Please, delete the proposition “Bovine mastitis is typically a result of inflammation in the mammary parenchyma, which is usually triggered by physical or microbial factors” and keep the next proposition-“Bovine mastitis is an inflammatory reaction of the mammary parenchyma that is usually caused by either physical or microbial factors [7]”.
-Linea 218:-Please correct CO2 in CO2
-Figures 1-6: Please modify the resolution of the figures, because is difficult to observe the finish line (migratory area) in your images, added a detailed title, and a legend with explanations of the abbreviations. Because the quality of your images is lower, you may have the option to move the figures to supplementary materials.
-Please add the figures with the cytotoxicity of your extracts against Vero cells and show the LC50 (mg/ml), and show the obtained results in a report with more references in the discussion chapter
-Please, add the limitations of your study or methods.
- Correct all grammatical errors, and write in vitro and in vivo by italic mode, in the entire manuscript.
-Please reformulate the conclusion to be according to the objective and results of the manuscript, and mention the detailed future direction of your study.
Minor editing of the English language required
Author Response
-In the introduction chapter, please more references about the cytotoxicity of the plant extracts to be in correlation with the manuscript objectives.
Response: This is a good point and justification for including cytotoxicity testing of the plant extracts has been added in to the Introduction (lines 103-108).
-Linea 46-47 –Please, delete the proposition “Bovine mastitis is typically a result of inflammation in the mammary parenchyma, which is usually triggered by physical or microbial factors” and keep the next proposition-“Bovine mastitis is an inflammatory reaction of the mammary parenchyma that is usually caused by either physical or microbial factors [7]”.
Response: Thank you for noting this repetition. The first sentence has been removed.
-Linea 218:-Please correct CO2 in CO2
Response: This has been corrected.
-Figures 1-6: Please modify the resolution of the figures, because is difficult to observe the finish line (migratory area) in your images, added a detailed title, and a legend with explanations of the abbreviations. Because the quality of your images is lower, you may have the option to move the figures to supplementary materials.
Response: We have attempted to improve the resolution of the figures as far as possible, and have included a more detailed title (i.e. “Note: AA, EA and RA represent acetone extracts of Antidesma venosum, Erythrina caffra and Searsia lancea leaves, while AB, EB and RB represent their ethanol extracts, respectively.”) for Figures 1 and 2, and the details remain the same for the rest of the figures. If this is still not satisfactory we will be happy to remove them to the Supplementary Material.
-Please add the figures with the cytotoxicity of your extracts against Vero cells and show the LC50 (mg/ml), and show the obtained results in a report with more references in the discussion chapter
Response: We carefully considered this comment and would like to leave the results as they are currently presented as there are several figures that would need to included, extending the paper. The figures will be available to interested readers upon request.
-Please, add the limitations of your study or methods.
Response: Limitations of the study have been included, together with future directions of research (section 4.5) in lines 173-185.
- Correct all grammatical errors, and write in vitro and in vivo by italic mode, in the entire manuscript.
Response: This has been corrected.
-Please reformulate the conclusion to be according to the objective and results of the manuscript, and mention the detailed future direction of your study.
Response: Thank you for this comment. The conclusion has been reformulated to accord more with the objective and results, and future directions of the study have been included in section 4.5, as well as in the conclusion.
The grammar and English language have also been revised.
Reviewer 4 Report
The manuscript described the evaluation of the antibacterial activities of nine plant extracts from South Africa against 12 Staphylococcus strains isolated from clinical bovine mastitis cases. Among them, eight were S. aureus, while the other four were non-aureus staphylococcus (NAS). The results indicated that Searsia lancea acetone extract exhibited the highest total antibacterial activities, while Erythrina caffra ethanol extract had the highest mean selectivity index. The observation of low cytotoxicity of the ethanol extracts of selected Antidesma venosum, S. lancea, and E. caffra made them become potent candidates for further studies. This study explored the applicability of nine localized plants against clinical drug-resistant S. aureus and non-aureus staphylococcus (NAS), suggesting the suitability of this study to be acceptable for publication in the journal “Applied Sciences.” Below are the suggestions for further improvement.
1. Abstract: The conclusion of this study should be described in the abstract to add the values.
2. Introduction: The reasons and the importance of choosing nine plants for this study should be described.
3. 3.2 Bacterial identification: The methods and the references for identifying phenotypic characteristics of bacterial isolates should be described. The results should be provided as supplementary materials.
4. 3.4 TLC and bioautography:
- The numbering of Figures 7 and 8 should be replaced with that of Figures 1 and 2. The numbering of other figures could be changed accordingly.
- The quality of TLC should be improved, and the clear zones on the TLC plates should be indicated and described.
- The artwork of Figures 1-8 could be improved. Figures 1-6 could be organized into one, while Figures 7-8 could be organized into another.
- The full name of the abbreviations, AA, EA, and RA, should be described for the first time in the text.
5. The arrangement of data in Tables 5, 6, 8, and 9 should be improved. It is better to show each value in the same row as in Table 7.
6. 3.7 Cytotoxicity and selectivity indices of plant extracts:
- The reasons for choosing three plants for cytotoxicity assays should be described at the beginning of this section.
- The selectivity index for the abbreviation SI should be shown here for the first time.
7. Conclusion:
- It only stated S. lancea exhibited the most potent antibacterial activity in this section, and it is suggested to describe all the solid results.
- The names of the ethanol extracts of the three plants should be described.
8. The format of the references should follow the rules of the journal “Applied Sciences”.
Only minor English language editing is required, which can be assisted by the editorial office.
Author Response
- Abstract: The conclusion of this study should be described in the abstract to add the values.
Response: Thank you very much for your observation. Changes have been made to the abstract to reflect your recommendation.
- Introduction: The reasons and the importance of choosing nine plants for this study should be described.
Response: Thank you for your suggestion. This has been included in the Materials and Methods, section 2.1
- 3.2 Bacterial identification: The methods and the references for identifying phenotypic characteristics of bacterial isolates should be described. The results should be provided as supplementary materials.
Response: The method was described in section 2.4. The results have been attached.
- 3.4 TLC and bioautography:
- The numbering of Figures 7 and 8 should be replaced with that of Figures 1 and 2. The numbering of other figures could be changed accordingly.
Response: This has been changed accordingly.
- The quality of TLC should be improved, and the clear zones on the TLC plates should be indicated and described.
Response: The clear zones have been marked out on the plates with black ink.
- The artwork of Figures 1-8 could be improved. Figures 1-6 could be organized into one, while Figures 7-8 could be organized into another.
Response: The artwork has been improved as far as possible.
- The full name of the abbreviations, AA, EA, and RA, should be described for the first time in the text.
Response: The full names of the abbreviations have been described.
- The arrangement of data in Tables 5, 6, 8, and 9 should be improved. It is better to show each value in the same row as in Table 7.
Response: Because of the inclusion of the standard error of the mean (SEM) and the number of columns in each of this table, it is challenging to have them in straight rows so we would prefer to retain them in their current format.
- 3.7 Cytotoxicity and selectivity indices of plant extracts:
- The reasons for choosing three plants for cytotoxicity assays should be described at the beginning of this section.
Response: Plants were chosen based on their MIC values below 0.1 mg/ml. This has been incorporated into this section.
- The selectivity index for the abbreviation SI should be shown here for the first time.
Response: Thank you for the suggestion. It has been done.
- Conclusion:
- It only stated S. lancea exhibited the most potent antibacterial activity in this section, and it is suggested to describe all the solid results.
Response: Other results have been incorporated into the conclusion.
- The names of the ethanol extracts of the three plants should be described.
Response: The names of the plants have been described at the beginning of this section.
- The format of the references should follow the rules of the journal “Applied Sciences”.
Response: The format of the references has been modified appropriately.
