The Isolation of a Novel Streptomyces sp. CJ13 from a Traditional Irish Folk Medicine Alkaline Grassland Soil that Inhibits Multiresistant Pathogens and Yeasts

Round 1

Reviewer 1 Report

Review of the article: “The isolation of a novel Streptomyces sp. CJ13 from a traditional Irish folk medicine alkaline grassland soil that inhibits multiresistant pathogens and yeasts.”

Submission ID applsci-1038191

I have read the manuscript with great interest. Because of the global spread of antibiotic resistant microorganisms, Gram-positive and Gram-negative bacteria but also pathogenic yeasts we urgently need new sources of not known to date antibiotics. I agree with opinion presented in introduction (lines47-56) that we have to change approach for research aiming in producing new antimicrobial agents – chemical modifications of known antibiotics is not sufficient we need completely new antibiotics of not known to date core structures.

Below I have presented my comments about manuscript. I would be grateful if the authors considered my remarks preparing final version of the article.

The most important advantages of the manuscript are:

1. Original idea of the research – isolation of new organisms - producers of antimicrobial agents from the “traditional medical soil” - alkaline grassland soil traditionally known for its healing properties from Boho, West Fermanagh (Ireland).

I would like to highlight the information presented between lines 86-90”However, respecting the strong sensitivities around this specific soil which remains within consecrated ground, we feel this cannot be considered as an exploitable resource for scientists in any normal sense. Instead, we returned to an adjacent upland alkaline grassland region which is still associated with traditional medicine approximately a kilometre from our original discovery to examine whether the same type of soil could be a new resource for antimicrobial discovery.”. I really respect and this decision and appreciate the respect for local traditions.

2. Advanced in silico research – identification of producing strain (Streptomyces CJ13), whole genome sequencing, phylogenetic analysis, prediction of gene clusters involved in production secondary metabolites and prediction of presence of antibiotic resistance genes.
3. Broad spectrum of activity of metabolite/metabolites produced by Streptomyces CJ13
4. The manuscript is prepared carefully, all experiments were well planned and performer and results are well documented and presented – the authors only should go through the whole text and check if names of microorganisms are written with italic (e.g. lines 175, 176, 177, 195, 196, 198, 199, 200, 209, 216 …)

The most important weaknesses of the manuscript

1. In contrast to in silico analysis only very preliminary research were performed in the scope of isolation and characterization of the active metabolite (metabolites?). There is information (lines 296-301) that authors have tried to use MS to identify the active constituents but they did not get interesting results – of course I understand it. However, some essential characteristic of active agents e.g., analysis of thermal and pH stability, resistance to the activity of proteases (it can be bacteriocins or antimicrobial peptides) would also be interesting. On the basis of the results presented in the manuscript we only know that the strain is able to produce “some” antimicrobial agent/agents.
2. It also would be interesting to check if the active metabolite is effectively produced in liquid medium – which medium is optimal for production.

Less important comments:

1. Please check the if names of bacteria are written with italic
2. Line 113 – “Sterilized washes of original soil” – I would be grateful for some more details.
3. Figure 3 - I think that in some cases the growth of Streptomyces sp. isolate CJ13 is visible on the test plates – particularly in the case of P. acnes. Am I wright? If yes it could suggest that this strain is able to grow in anaerobic conditions, which is not in agreement with information presented in Table 1.
4. Why there are so important differences in activity of light and dark variant - both of them are Streptomyces sp. isolate CJ13

Summarizing interesting strain has been isolated. Advanced in silico analysis has been performed and only very preliminary characteristic of produced agent has been presented (in fact only broad of spectrum). However, my final opinion is positive. I believe that the authors will continue these research and will be able to produce new potential antibiotic highly efficient against broad spectrum of pathogenic bacteria and yeasts.

Final decision – minor revision.

Author Response

Please see the attachment

Author Response File: Author Response.docx

Reviewer 2 Report

In the research article the authors describe an isolate of Streptomyces sp. and its antibacterial and antifungal activity. The authors focused in their work on the isolation of Streptomyces from soil samples of Northern Ireland. The activity of the Streptomyces sp isolate CJ13, was determined toward multiresistant bacterial and yeast strains. Further studies  involved phylogenetic analysis and whole genome sequence analysis with antiSMASH software to predict the potential for the synthesis of secondary metabolite clusters. Overall, the manuscript is clearly presented, however some sections require additional information. I have the following comments for the authors regarding their study:

• The authors did not specify in their article the basis for the identification of the isolate as Streptomyces, was it based only on the characterization of morphological features? This requires some further clarification.
• The antibacterial activity requires further explanation and additional information since this is the main focus of this research study. It would be beneficial to present the results of the agar diffusion assay additionally in the form of a Table with the provided measurements of the zones of inhibition for better comparison of the obtained results. Additional information regarding the technical  aspects of this assay are also required. The Materials and Methods section requires supplementation with information regarding the application of Streptomyces in the agar diffusion assay. The authors briefly mention that the agar cores with Streptomyces were used in this assay. However, some quantitative information would be required. In what form was the isolate added to the wells, was the isolate applied in the form of an extract and in what concentration? Furthermore, the authors in their study provide only preliminary results regarding the activity of the isolate, as determined by the agar diffusion assay. The determination of MIC values would be more informative.
• Some errors are present in the Figure 3 description. The abbreviations in the description of the Figure should be same as those used in the image. Please provide the calculated final concentration of chlorhexidine in the description.

Author Response

Please see the attachment

Round 2

Reviewer 2 Report

The authors have added the requested information to the manuscript and made appropriate changes accordingly.