<xhtml:span xmlns:xhtml="http://www.w3.org/1999/xhtml" xml:lang="en">Effect of Antibiotic Administration on Blastocystis Persistence and Gut Microbiome-Metabolome Dynamics in an Irritable Bowel Syndrome Longitudinal Case Study </xhtml:span>
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Background: Blastocystis, the most prevalent microbial eukaryote in humans, has a global distribution. Studies have linked its presence with distinct gut microbiome and metabolome profiles compared to those where the organism is absent. However, the interplay of antibiotics administration, Blastocystis and the surrounding gut microbiome remains understudied. This case study aimed to explore antibiotic consumption and the presence of Blastocystis with subsequent changes in the gut microbiome and metabolome of an individual diagnosed with irritable bowel syndrome (IBS). Methods: Stool samples from an IBS patient, collected at 12 time points, were tested for Blastocystis presence using RT-PCR targeting the SSUrRNA gene, followed by sequencing of positive samples. Illumina sequencing determined the gut microbiome composition, while one-dimensional proton NMR spectroscopy was used to analyse the metabolome composition. Statistical analyses were conducted to identify relationships between antibiotic consumption, bacterial diversity, metabolome composition, and Blastocystis presence. Results: Antibiotics significantly impacted the gut microbiome, with diversity declining early in the antibiotic course, then recovering later and post-course. Blastocystis was detected early, late, and post-course but was not detectable mid-course, coinciding with the decline in bacterial diversity. Significant differences were observed between Blastocystis-positive and Blastocystis-negative samples with bacterial composition significantly changing between samples collected before, early, and after the antibiotic course compared to those collected mid-course. Metabolite groups, including short-chain fatty acids, amino acids, and succinate, exhibited changes throughout the antibiotic course, indicating that gut metabolite composition is affected by antibiotic consumption. Discussion/Conclusion: While antibiotics did not significantly impact Blastocystis colonisation, they did cause a mid-course decline in microbial diversity and Blastocystis presence. The study also revealed significant alterations in important metabolites such as SCFAs and amino acids throughout the antibiotic course, with an altered metabolome observed post-course. This case study underscores the complex interactions between antibiotics, gut microbiota, and metabolites, highlighting the resilience of Blastocystis in the gut ecosystem.
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Thank you for the privilege of reviewing your work and apologies for the delay. The reviewers find your work of interest but have suggested major revisions before this work can be accepted for publication in Access Microbiology. I would like to invite you to re-submit a revised version of your manuscript that addresses the reviewer’s comments, most notably centred on the use of euclidean distance for compositional data analyses and only 1 IBS patient (n=1). Please return the manuscript within 50 days; if you cannot complete the modification within this time period, please contact me. If you do not wish to modify the manuscript and prefer to submit it to another journal, notify me immediately so that the manuscript may be formally withdrawn from consideration at Access Microbiology. Please note the following requirement for …
Thank you for the privilege of reviewing your work and apologies for the delay. The reviewers find your work of interest but have suggested major revisions before this work can be accepted for publication in Access Microbiology. I would like to invite you to re-submit a revised version of your manuscript that addresses the reviewer’s comments, most notably centred on the use of euclidean distance for compositional data analyses and only 1 IBS patient (n=1). Please return the manuscript within 50 days; if you cannot complete the modification within this time period, please contact me. If you do not wish to modify the manuscript and prefer to submit it to another journal, notify me immediately so that the manuscript may be formally withdrawn from consideration at Access Microbiology. Please note the following requirement for re-submitting your revised manuscript: • Upload point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT in your cover letter.
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Comments to Author
The manuscript by Newton et al presents an interesting case study exploring the impact of antibiotics on Blastocystis colonisation, gut microbiome composition, and metabolome dynamics in an IBS patient. The integration of microbiome and metabolome analysis provides valuable insights into the potential resilience and interaction of Blastocystis within the gut ecosystem, particularly during antibiotic exposure. Overall, the manuscript is generally well-structured, with clear descriptions of methods, results and integrated discussion, and final conclusion. However, this reviewer has a few suggestions to be considered by the authors to potentially enhance the case study findings. * The manuscript would benefit from further statistical analysis. Although this is only an n = 1, additional details on effect …
Comments to Author
The manuscript by Newton et al presents an interesting case study exploring the impact of antibiotics on Blastocystis colonisation, gut microbiome composition, and metabolome dynamics in an IBS patient. The integration of microbiome and metabolome analysis provides valuable insights into the potential resilience and interaction of Blastocystis within the gut ecosystem, particularly during antibiotic exposure. Overall, the manuscript is generally well-structured, with clear descriptions of methods, results and integrated discussion, and final conclusion. However, this reviewer has a few suggestions to be considered by the authors to potentially enhance the case study findings. * The manuscript would benefit from further statistical analysis. Although this is only an n = 1, additional details on effect sizes and specific comparisons between time points may add further clarity to the findings. * It would also be of interest to integrate the microbiota, Blastocystis and metabolomics data to provide a more in-depth picture of potential functional interactions. * While this is a case study, the authors should acknowledge the limitations regarding sample size and the ability to generalise findings. This could be addressed in more detail in the final conclusion section, emphasising the very exploratory nature and the need for larger cohort studies to confirm these observations. * The manuscript suggests that antibiotics significantly impacted the microbiome, but there is limited discussion on how specific antibiotics used (amoxicillin and clarithromycin) may differentially influence gut bacteria. Some literature on these antibiotics' effects on gut microbial diversity and specific taxa would strengthen this particular section. This should be backed up by the addition of relevant references as appropriate. * Some figures, particularly the heatmaps and compositional plots, could be refined for clarity. In certain cases, taxa labels are hard to read, and additional annotations on significant findings in these figures would make them more interpretable. However this reviewer appreciates this may be due to the quality of the PDF provided for review. * The manuscript hypothesises that Blastocystis may play an active role as a "gut ecosystem engineer". While intriguing, this claim could benefit from further discussion and support from previous literature, especially studies on microbial interactions that might explain Blastocystis role in microbiome stability or diversity. * There are some minor typographical errors throughout the manuscript that should be corrected in the revised manuscript version.
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Very good
To what extent are the conclusions supported by the data?
Strongly 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
The paper is well written and very concise. Overall, the authors did a good job of framing their work and providing a detailed description of their methods. I think this pilot study is well planned and I think it is quite an understudied subject. However, I have some concerns listed below mainly on the analysis front. Major comments - The use of PCA(read: euclidean distance) is generally not recommended for microbiome analysis. It has been well documented within field (10.1101/gr.085464.108, https://doi.org/10.1038/nmeth.1499). A better alternative would be Bray Curtis with a supplementary of Unifrac (if phylogeny is import) or Jaccard (if presence/absence is the most import thing). - Table 1 - Is the presence of Bastocystis determined by qPCR? I can't find information to what the defining criteria …
Comments to Author
The paper is well written and very concise. Overall, the authors did a good job of framing their work and providing a detailed description of their methods. I think this pilot study is well planned and I think it is quite an understudied subject. However, I have some concerns listed below mainly on the analysis front. Major comments - The use of PCA(read: euclidean distance) is generally not recommended for microbiome analysis. It has been well documented within field (10.1101/gr.085464.108, https://doi.org/10.1038/nmeth.1499). A better alternative would be Bray Curtis with a supplementary of Unifrac (if phylogeny is import) or Jaccard (if presence/absence is the most import thing). - Table 1 - Is the presence of Bastocystis determined by qPCR? I can't find information to what the defining criteria of how the authors define "positive" and "negative". - I would recommend including an ordination plot included. It will be much better indication of how similar the gut microbiome is with/without Blastocystis. - Metabolite composition can benefit from having an ordination plot to highlight observed in different conditions. Here, a PCA plot would be helpful. - The metabolomic section lacks a comprehensive analysis. All of the description of the results are observational. The authors can benefit by including statistical testing to show that the differences observe ties in with the treatment. - A potential missed opportunity is linking the metabolomic analysis with the gut microbiome dataset. Is the changes in the metabolite a result of the treatment, changes in the bacterial species in the gut, or other factors? - The authors concluded that there are metabolite changes due to the use of antibiotic. I'm not sure if that is the right conclusion given the authors have not linked the gut microbiome to the metabolomic analysis. Minor comments line 83 - Either the () is missing information or it is a typo. line 162 - Another [] with either missing ifnormation or a typo. Line 182 - Include the version of R packages used for the analysis.
Please rate the manuscript for methodological rigour
Poor
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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