Development of selective isolation media for detecting Actinomyces and Schaalia genera from oral specimens containing indigenous bacteria
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To isolate specific bacteria from samples constituting the microbiota, it is essential to employ selective media that suppress the growth of resident bacteria other than specific target bacteria. Selective media for clinically important Actinomyces (including Schaalia, which was previously taxonomically classified as part of the Actinomyces genus) have been limited because they have been designed for a limited range of species within the genus and require ingredients which are difficult to prepare and handle. This study aimed to develop a selective medium (referred to as Actinomyces and Schaalia Selective Medium (ASSM)) for the isolation of a broad range of Actinomyces and Schaalia species from samples mixed with resident bacteria. The composition of ASSM includes yeast extract, agar, brain heart infusion (BHI), levofloxacin (LVFX), fosfomycin (FOM), colistin (CL), and metronidazole (MNZ). Evaluation of the medium using 24 swab samples serially collected from the roots of the teeth of a healthy individual for whom metagenome sequencing data of a saliva sample are publicly available revealed that ASSM adjusted to concentrations of LVFX 0.5 mg/L, FOM 5 mg/L, CL 1 mg/L, and MNZ 2 mg/L and cultured anaerobically at 35°C for 7 days enabled the isolation of Actinomyces species from 37.5% of the samples. The inclusion of CL and MNZ in ASSM can also be useful for samples suspected of harbouring other bacterial species. The selective isolation medium is expected to contribute to studies investigating the relationship between these bacteria and their pathogenesis or disease.
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Thank you for your careful consideration of the comments by the reviewers and addressing these satisfactorily.
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Please make sure that you clarify your method section a bit more, it would also be helpful if you explained on which criteria the reference strains were chosen, that would improve clarity and readibility. I support the comment about the flowchart on the methodolgy from reviewer 1. I think if indeed only one patient was sampled, this should be raised in the discussion as a potential limitation and that this study could be seen as an indicator. I understand that possibly there was only this one patient available with the metagenomics data available as well? I was unable to find the accession number for the metagenome in the manuscript, could you please provide this and also the reference to the corresponding publication? It seems that 4 samples were taken at each time point? Are you showing the mean values? Could you explain why these …
Please make sure that you clarify your method section a bit more, it would also be helpful if you explained on which criteria the reference strains were chosen, that would improve clarity and readibility. I support the comment about the flowchart on the methodolgy from reviewer 1. I think if indeed only one patient was sampled, this should be raised in the discussion as a potential limitation and that this study could be seen as an indicator. I understand that possibly there was only this one patient available with the metagenomics data available as well? I was unable to find the accession number for the metagenome in the manuscript, could you please provide this and also the reference to the corresponding publication? It seems that 4 samples were taken at each time point? Are you showing the mean values? Could you explain why these timepoints were chosen? How many replicates did you carry out for each of the reference strains? This is not clear to me from the current version. It is not clear what Organism 1,2,3 etc refer to in Table 1, please include a legend that decodes this or name the organisms there.
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Comments to Author
Dear author, I read your work carefully with great interest. Please find below a couple of comments and suggestions. - General : The articles is short but clear and well-referenced. The purpose of the study is quickly understood. - M&M : Need a little clarification Line 75-83 :Reference and clinical strains Please specify the origin of the clinical strains mentioned "previously" in table 4 (perhaps ref 10?). "The clinical strains used in this study were isolated in a previous study (ref X)." Line 80 clinical strain instead of clinical stain - Results : just one comment The results obtained without carefully reading the M&Ms are quite difficult to understand at first. Especially when it comes to recovery rates (above 100). Perhaps indicating raw data? - Discussion : just one comment Line 157-163 …
Comments to Author
Dear author, I read your work carefully with great interest. Please find below a couple of comments and suggestions. - General : The articles is short but clear and well-referenced. The purpose of the study is quickly understood. - M&M : Need a little clarification Line 75-83 :Reference and clinical strains Please specify the origin of the clinical strains mentioned "previously" in table 4 (perhaps ref 10?). "The clinical strains used in this study were isolated in a previous study (ref X)." Line 80 clinical strain instead of clinical stain - Results : just one comment The results obtained without carefully reading the M&Ms are quite difficult to understand at first. Especially when it comes to recovery rates (above 100). Perhaps indicating raw data? - Discussion : just one comment Line 157-163 This section can also be inserted in the introduction? - References : just one correction Ref 10: please correct line 317-318: duplication of author list instead of article title: "Isolation and Identification Methods for Actinomyces israelii Involved in Actinomycosis".
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
Although the presented research is of current relevance as a wide range of selective media would improve identification and efficacy of isolation when one wants to study specific strains, the research needs further clarifications. 1. At times the document flow and reasoning behind the methods used are not very clear. I would go over the document, improve flow and clarity. Adding a flowchart to highlights/summarise methods used would also improve readability 2. Number of clinical samples tested: Only testing 1 clinical sample is not enough to prove that a new medium is more selective than a conventional medium. What is the power of your current experiment? This needs to be worked out to identify how many samples need to be tested to compare the conventional medium with the new medium 3. Statistical …
Comments to Author
Although the presented research is of current relevance as a wide range of selective media would improve identification and efficacy of isolation when one wants to study specific strains, the research needs further clarifications. 1. At times the document flow and reasoning behind the methods used are not very clear. I would go over the document, improve flow and clarity. Adding a flowchart to highlights/summarise methods used would also improve readability 2. Number of clinical samples tested: Only testing 1 clinical sample is not enough to prove that a new medium is more selective than a conventional medium. What is the power of your current experiment? This needs to be worked out to identify how many samples need to be tested to compare the conventional medium with the new medium 3. Statistical analysis: Throughout the document there is no mention of replicates and statistical analysis to see if there is any statistically significant difference between the conventional agar/medium vs the proposed medium/agar. 4. Ethical approval for sample collection: Please highlight this in the document
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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