Statistical Medium Optimization for the Production of Anti-Methicillin-Resistant Staphylococcus aureus Metabolites from a Coal-Mining-Soil-Derived Streptomyces rochei CMB47
and Ines Mancini 2,*
Round 1
Reviewer 1 Report
The paper entitled “Statistical Medium Optimization for the Production of anti- Methicillin-Resistant Staphylococcus aureus Metabolites from a Coal Mining Soil Derived Streptomyces rochei CMB47” written by Ibtissem Djinni and co-workers described the isolation and culture optimization of a coal mining soil derived Streptomyces rochei CMB47, along with the isolation and preliminary characterization of anti-MRSA secondary metabolites from its fermentation products. These findings will support the potential interest in a further investigation on the discovery of the anti-MRSA metabolites. The languages are suggested to be improved while some descriptions are tediously long. Moreover, there are some points as shown below that could be greatly addressed to further improve the manuscript, which can be accepted in fermentation after major revision.
Some points:
1. Since there were three compounds detected in the bioactive fraction 11, it is strongly suggested to carry out the isolation, structural characterization, and bioactive test of them.
2. Some figures are suggested to be provided in the supporting information, such as figures 5 and 6.
3. Some words or descriptions should be italic. Such as Line 252, v/v; Lines 261, 474, tR; Line 324, Streptomyces; Lines 331, 333, 484, Streptomyces rochei; Line 351, E. coli; Lines 459, 462, 475, m/z; Lines 474, 479, S. rochei;
Author Response
We are very grateful to the reviewer for the time devoted to the revision, as well as for the valuable feedback and constructive comments on the manuscript.
The changes are currently highlighted within the new version of the manuscript. Please find below, point-by-point responses to the specific comments and suggestions of the reviewers.
The paper entitled “Statistical Medium Optimization for the Production of anti- Methicillin-Resistant Staphylococcus aureus Metabolites from a Coal Mining Soil Derived Streptomyces rochei CMB47” written by Ibtissem Djinni and co-workers described the isolation and culture optimization of a coal mining soil derived Streptomyces rochei CMB47, along with the isolation and preliminary characterization of anti-MRSA secondary metabolites from its fermentation products. These findings will support the potential interest in a further investigation on the discovery of the anti-MRSA metabolites. The languages are suggested to be improved while some descriptions are tediously long.
From the authors:
The language has been improved. We agree with the reviewer that some parts are wordy, but the detailed description must be given in order to characterize the specificity of the strain, especially when comparing different strains.
Moreover, there are some points as shown below that could be greatly addressed to further improve the manuscript, which can be accepted in fermentation after major revision.
Some points:
- Since there were three compounds detected in the bioactive fraction 11, it is strongly suggested to carry out the isolation, structural characterization, and bioactive test of them.
Answer from the authors:
By HPLC-DAD-ESI-MS analysis, we identified three compounds having molecular masses of 206, 220 and 234 Da, having very similar molecular structures , as based on the qualitative UV spectrum acquired for each of them. Furthermore, as evident in Figure 6b (moved in the Supplementary as required by the reviewer), the compound eluted at 8.2 min, having a molecular mass =220 Da, is clearly the most abundant of the three compounds present in the fraction 11B. Therefore, the anti-MRSA activity evaluated for this fraction (showing the highest activity against MRSA, among the tested fractions, with inhibitory zones of 35mm in comparison to 19 mm of vancomycin (30µg/disc), as indicated in paragraph 3.6) is mainly attributable to this metabolite, assuming that compounds with similar structures should have comparable bioactivity.
This is what can be speculated based on the data available so far.
Nevertheless, the comparison of molecular mass values obtained for the three metabolites produced by S. rochei CMB47 with all known antibiotics produced by S. rochei support their novel molecular structures. This chemical elucidation requires additional extensive analyses (probably on an increased amount of sample that we may need to produce) and the results will be published in a separate paper.
This second consideration has been introduced in Conclusions in the following form:”These results warrant further investigation on the structural elucidation of the bioactive metabolites, the molecular mass values of which, to the best of our knowledge, have not found any correspondence with those of antibiotic metabolites isolated so far from S. rochei.”
- Some figures are suggested to be provided in the supporting information, such as figures 5 and 6.
Answer from the authors:
The figures were moved to Supplementary, as Figures S1 and S2, respectively.
- Some words or descriptions should be italic. Such as Line 252, v/v; Lines 261, 474, tR; Line 324, Streptomyces; Lines 331, 333, 484, Streptomyces rochei; Line 351, E. coli; Lines 459, 462, 475, m/z; Lines 474, 479, S. rochei;
Answer from the authors: done
- Introduction must be improved
Answer from the authors:
Regarding the topics illustrated in the Introduction, we think that the right points were addressed making the state of the art of this study on the investigation for new antibacterial metabolites directed against the S. aureus and its resistant MRSA form. The emergence of drug resistant pathogens reported in the WHO list highlights the aims of this ambitious but overly necessary study, in the search for new drugs due to the ineffectiveness of current treatments and a renewed market demand.
In particular, the investigated actinobacteria strain described in this work derived from a particular less-studied ecosystem and microorganisms isolated from these habitats have been
shown to be promising sources of biologically active metabolites characterized by peculiar molecular structures. Additionally, the fermentation conditions for an improved production of active metabolites was stated by applying statistical approaches.
At line 45 the sentence “Secondary metabolites produced by microorganisms, specifically Actinobacteria, are regarded one of the major sources of antibacterial agents ” was introduced to correlate more properly the topics .
The Introduction was improved in its English form.
- Is the research design appropriate?
Answer from the authors:
We think that the research design is appropriated for the biological part of this work, based on the following considerations and results:
- The isolation of actinobacteria from peculiar natural habitats, as the Algerian coil mine of this case, is of interest to avoid re-isolation of strains that produce known bioactive metabolites and usually lead to highly diverse actinobacterial communities.
- CMB47 isolate was selected for its significant antagonistic potential against MRSA and aureus. The strain was identified based on polyphasic and molecular characterization, as well as on the study of the metabolites production kinetic against MRSA.
- The metabolic profile of active isolate was identified, and the modeling and optimization of the bioactive compounds production using statistical approach was performed by rotatable central composite design and response surface methodology.
- bioassay guided fractionation of the active crude extract allowed to identify the fraction containing the active metabolites
- HPLC-DAD/ESIMS analysis provided very preliminary structural indications on the compounds responsible for MRSA inhibition and an extensive chemical study will be necessary for establishing the metabolites, emerging as new structures based on the molecular mass identified so far.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments on Manuscript fermentation-2333885
General Comments
The authors have made intensive investigations of strain Steptomyces rochei CMB47, especially its taxonomic characterization by morphologic properties, genomic DNA extraction, 16S rRNA gene and phylogenetic analyis, extracellular enzymes production, medium and cultivation optimization for growth and increased biological activity against methicillin-resistant Staphylococcus aureus, and the preliminary characterization of the biological activity produced by S. rochei CMB47. The authors identified three compounds by HPLC-DAD-ESI-MS analysis having molecular masses of 206, 220 and 234 Da. Nevertheless, such an intensive methodological procedure is justified when the chemical nature of the produced biological active metabolites is novel or seems to be novel. There is missing the comparison of the three metabolites produced by S. rochei CMB47 regarding their molecular masses with all known antibiotics produced by S. rochei. It is strongly recommended to publish the chemical data of the three metabolites in a separate paper as soon as possible to prove their novelty, that allows in additon their quantification in the fermentation process.
The manuscript is well written and can be accepted for publication after some corrections considering the above comments.
Specific Comments
Lines 23 and 356: A value behind the comma makes no sense in the case of inhibitory zones.
Line 87: Change to ‘preliminary new metabolites‘.
Lines 324, 331, 333, 334, 339, 350, 474, 479, 484, 488: Streptomyces and Streptomyces rochei should be written in italics.
Author Response
We are very grateful to the reviewer for the time devoted to the revision, as well as for the valuable feedback and constructive comments on the manuscript.
The changes are currently highlighted within the new version of the manuscript. Please find below, point-by-point responses to the specific comments and suggestions of the reviewers.
General Comments
The authors have made intensive investigations of strain Steptomyces rochei CMB47, especially its taxonomic characterization by morphologic properties, genomic DNA extraction, 16S rRNA gene and phylogenetic analyis, extracellular enzymes production, medium and cultivation optimization for growth and increased biological activity against methicillin-resistant Staphylococcus aureus, and the preliminary characterization of the biological activity produced by S. rochei CMB47.
The authors identified three compounds by HPLC-DAD-ESI-MS analysis having molecular masses of 206, 220 and 234 Da. Nevertheless, such an intensive methodological procedure is justified when the chemical nature of the produced biological active metabolites is novel or
seems to be novel. There is missing the comparison of the three metabolites produced by S. rochei CMB47 regarding their molecular masses with all known antibiotics produced by S. rochei. It is strongly recommended to publish the chemical data of the three metabolites in a separate paper as soon as possible to prove their novelty, that allows in additon their quantification in the fermentation process.
Answer from the authors:
At line 473 this sentence was introduced: “No correlations was found by the comparison of molecular masses of the three metabolites produced by S. rochei CMB47 with all known antibiotics produced by S. rochei, supporting the potential novelty of their molecular structures.” Additionally, the conclusions already indicated: “These results warrant further investigation on the structural elucidation of the bioactive metabolites, the molecular mass values of which, to the best of our knowledge, have not found any correspondence with those of antibiotic metabolites isolated so far from S. rochei.”
The manuscript is well written and can be accepted for publication after some corrections considering the above comments.
Specific Comments
Lines 23 and 356: A value behind the comma makes no sense in the case of inhibitory zones.
Answer from the authors:
We thank the reviewer for this comment and have modified the experimental values accordingly, whereas we preserve the original values derived from model calculation reported in Table S1. This is true for y (diameter of inhibition zones) which were used for the model elaboration.
Line 87: Change to ‘preliminary new metabolites‘.
Answer from the authors: done
Lines 324, 331, 333, 334, 339, 350, 474, 479, 484, 488: Streptomyces and Streptomyces rochei should be written in italics.
Answer from the authors: done
Regarding the comment “the research design can be improved”:
Answer from the authors:
We think that the research design is appropriated for the biological part of this work, based on the following considerations and results:
- a) The isolation of actinobacteria from peculair natural habitats, as the Algerian coil mine of this case, is of interest to avoid re-isolation of strains that produce known bioactive metabolites and usually lead to highly diverse actinobacterial communities.
- b) CMB47 isolate was selected for its significant antagonistic potential against MRSA and S. aureus. The strain was identified based on polyphasic and molecular characterization, as well as on the study of the metabolites production kinetic against MRSA.
- c) The metabolic profile of active isolate was identified, and the modeling and optimization of the bioactive compounds production using statistical approach was performed by rotatable central composite design and response surface methodology.
- d) bioassay guided fractionation of the active crude extract allowed to identify the fraction containing the active metabolites
- e) HPLC-DAD/ESIMS analysis provided very preliminary structural indications on the compounds responsible for MRSA inhibition and an extensive chemical study will be necessary for establishing the metabolite, emerging as new structures based on the molecular mass identified so far.
At the end of paragraph 3.6, a detail has been introduced on the preliminary indications for molecular metabolites responsible of bioactivity by the following sentence: :
“No correlations was found by the comparison of molecular masses of the three metabolites produced by S. rochei CMB47 with all known antibiotics produced by S. rochei (cited in Introduction) supporting the potential novelty of their molecular structures. “
Regarding the comment “the conclusions supported by the results can be improved”:
Answer from the authors:
The original sentence has been implemented as follows:
“These results warrant further investigation on the structural elucidation of the bioactive metabolites, the molecular mass values of which, to the best of our knowledge, have not found any correspondence with those of antibiotic metabolites isolated so far from S. rochei.”
We are very grateful to the reviewer for the time devoted to the revision, as well as for the valuable feedback and constructive comments on the manuscript.
The changes are currently highlighted within the new version of the manuscript. Please find below, point-by-point responses to the specific comments and suggestions of the reviewers.
General Comments
The authors have made intensive investigations of strain Steptomyces rochei CMB47, especially its taxonomic characterization by morphologic properties, genomic DNA extraction, 16S rRNA gene and phylogenetic analyis, extracellular enzymes production, medium and cultivation optimization for growth and increased biological activity against methicillin-resistant Staphylococcus aureus, and the preliminary characterization of the biological activity produced by S. rochei CMB47.
The authors identified three compounds by HPLC-DAD-ESI-MS analysis having molecular masses of 206, 220 and 234 Da. Nevertheless, such an intensive methodological procedure is justified when the chemical nature of the produced biological active metabolites is novel or
seems to be novel. There is missing the comparison of the three metabolites produced by S. rochei CMB47 regarding their molecular masses with all known antibiotics produced by S. rochei. It is strongly recommended to publish the chemical data of the three metabolites in a separate paper as soon as possible to prove their novelty, that allows in additon their quantification in the fermentation process.
Answer from the authors:
At line 473 this sentence was introduced: “No correlations was found by the comparison of molecular masses of the three metabolites produced by S. rochei CMB47 with all known antibiotics produced by S. rochei, supporting the potential novelty of their molecular structures.” Additionally, the conclusions already indicated: “These results warrant further investigation on the structural elucidation of the bioactive metabolites, the molecular mass values of which, to the best of our knowledge, have not found any correspondence with those of antibiotic metabolites isolated so far from S. rochei.”
The manuscript is well written and can be accepted for publication after some corrections considering the above comments.
Specific Comments
Lines 23 and 356: A value behind the comma makes no sense in the case of inhibitory zones.
Answer from the authors:
We thank the reviewer for this comment and have modified the experimental values accordingly, whereas we preserve the original values derived from model calculation reported in Table S1. This is true for y (diameter of inhibition zones) which were used for the model elaboration.
Line 87: Change to ‘preliminary new metabolites‘.
Answer from the authors: done
Lines 324, 331, 333, 334, 339, 350, 474, 479, 484, 488: Streptomyces and Streptomyces rochei should be written in italics.
Answer from the authors: done
Regarding the comment “the research design can be improved”:
Answer from the authors:
We think that the research design is appropriated for the biological part of this work, based on the following considerations and results:
- a) The isolation of actinobacteria from peculair natural habitats, as the Algerian coil mine of this case, is of interest to avoid re-isolation of strains that produce known bioactive metabolites and usually lead to highly diverse actinobacterial communities.
- b) CMB47 isolate was selected for its significant antagonistic potential against MRSA and S. aureus. The strain was identified based on polyphasic and molecular characterization, as well as on the study of the metabolites production kinetic against MRSA.
- c) The metabolic profile of active isolate was identified, and the modeling and optimization of the bioactive compounds production using statistical approach was performed by rotatable central composite design and response surface methodology.
- d) bioassay guided fractionation of the active crude extract allowed to identify the fraction containing the active metabolites
- e) HPLC-DAD/ESIMS analysis provided very preliminary structural indications on the compounds responsible for MRSA inhibition and an extensive chemical study will be necessary for establishing the metabolite, emerging as new structures based on the molecular mass identified so far.
At the end of paragraph 3.6, a detail has been introduced on the preliminary indications for molecular metabolites responsible of bioactivity by the following sentence: :
“No correlations was found by the comparison of molecular masses of the three metabolites produced by S. rochei CMB47 with all known antibiotics produced by S. rochei (cited in Introduction) supporting the potential novelty of their molecular structures. “
Regarding the comment “the conclusions supported by the results can be improved”:
Answer from the authors:
The original sentence has been implemented as follows:
“These results warrant further investigation on the structural elucidation of the bioactive metabolites, the molecular mass values of which, to the best of our knowledge, have not found any correspondence with those of antibiotic metabolites isolated so far from S. rochei.”
We are very grateful to the reviewer for the time devoted to the revision, as well as for the valuable feedback and constructive comments on the manuscript.
The changes are currently highlighted within the new version of the manuscript. Please find below, point-by-point responses to the specific comments and suggestions of the reviewers.
General Comments
The authors have made intensive investigations of strain Steptomyces rochei CMB47, especially its taxonomic characterization by morphologic properties, genomic DNA extraction, 16S rRNA gene and phylogenetic analyis, extracellular enzymes production, medium and cultivation optimization for growth and increased biological activity against methicillin-resistant Staphylococcus aureus, and the preliminary characterization of the biological activity produced by S. rochei CMB47.
The authors identified three compounds by HPLC-DAD-ESI-MS analysis having molecular masses of 206, 220 and 234 Da. Nevertheless, such an intensive methodological procedure is justified when the chemical nature of the produced biological active metabolites is novel or
seems to be novel. There is missing the comparison of the three metabolites produced by S. rochei CMB47 regarding their molecular masses with all known antibiotics produced by S. rochei. It is strongly recommended to publish the chemical data of the three metabolites in a separate paper as soon as possible to prove their novelty, that allows in additon their quantification in the fermentation process.
Answer from the authors:
At line 473 this sentence was introduced: “No correlations was found by the comparison of molecular masses of the three metabolites produced by S. rochei CMB47 with all known antibiotics produced by S. rochei, supporting the potential novelty of their molecular structures.” Additionally, the conclusions already indicated: “These results warrant further investigation on the structural elucidation of the bioactive metabolites, the molecular mass values of which, to the best of our knowledge, have not found any correspondence with those of antibiotic metabolites isolated so far from S. rochei.”
The manuscript is well written and can be accepted for publication after some corrections considering the above comments.
Specific Comments
Lines 23 and 356: A value behind the comma makes no sense in the case of inhibitory zones.
Answer from the authors:
We thank the reviewer for this comment and have modified the experimental values accordingly, whereas we preserve the original values derived from model calculation reported in Table S1. This is true for y (diameter of inhibition zones) which were used for the model elaboration.
Line 87: Change to ‘preliminary new metabolites‘.
Answer from the authors: done
Lines 324, 331, 333, 334, 339, 350, 474, 479, 484, 488: Streptomyces and Streptomyces rochei should be written in italics.
Answer from the authors: done
Regarding the comment “the research design can be improved”:
Answer from the authors:
We think that the research design is appropriated for the biological part of this work, based on the following considerations and results:
- a) The isolation of actinobacteria from peculair natural habitats, as the Algerian coil mine of this case, is of interest to avoid re-isolation of strains that produce known bioactive metabolites and usually lead to highly diverse actinobacterial communities.
- b) CMB47 isolate was selected for its significant antagonistic potential against MRSA and S. aureus. The strain was identified based on polyphasic and molecular characterization, as well as on the study of the metabolites production kinetic against MRSA.
- c) The metabolic profile of active isolate was identified, and the modeling and optimization of the bioactive compounds production using statistical approach was performed by rotatable central composite design and response surface methodology.
- d) bioassay guided fractionation of the active crude extract allowed to identify the fraction containing the active metabolites
- e) HPLC-DAD/ESIMS analysis provided very preliminary structural indications on the compounds responsible for MRSA inhibition and an extensive chemical study will be necessary for establishing the metabolite, emerging as new structures based on the molecular mass identified so far.
At the end of paragraph 3.6, a detail has been introduced on the preliminary indications for molecular metabolites responsible of bioactivity by the following sentence: :
“No correlations was found by the comparison of molecular masses of the three metabolites produced by S. rochei CMB47 with all known antibiotics produced by S. rochei (cited in Introduction) supporting the potential novelty of their molecular structures. “
Regarding the comment “the conclusions supported by the results can be improved”:
Answer from the authors:
The original sentence has been implemented as follows:
“These results warrant further investigation on the structural elucidation of the bioactive metabolites, the molecular mass values of which, to the best of our knowledge, have not found any correspondence with those of antibiotic metabolites isolated so far from S. rochei.”
Author Response File: Author Response.pdf
