Cerebrospinal Fluid from Healthy Pregnant Women Does Not Harbor a Detectable Microbial Community

ABSTRACT Cerebrospinal fluid (CSF) circulating in the human central nervous system has long been considered aseptic in healthy individuals, because normally, the blood-brain barrier can protect against microbial invasions. However, this dogma has been called into question by several reports that microbes were identified in human brains, raising the question of whether there is a microbial community in the CSF of healthy individuals without neurological diseases. Here, we collected CSF samples and other samples, including one-to-one matched oral and skin swab samples (positive controls), from 23 pregnant women aged between 23 and 40 years. Normal saline samples (negative controls), sterile swabs, and extraction buffer samples (contamination controls) were also collected. Twelve of the CSF specimens were also used to evaluate the physiological activities of detected microbes. Metagenomic and metatranscriptomic sequencing was performed in these 116 specimens. A total of 620 nonredundant microbes were detected, which were dominated by bacteria (74.6%) and viruses (24.2%), while in CSF samples, metagenomic sequencing found only 26 nonredundant microbes, including one eukaryote, four bacteria, and 21 viruses (mostly bacteriophages). The beta diversity of microbes compared between CSF metagenomic samples and other types of samples (except negative controls) was significantly different from that of the CSF self-comparison. In addition, there was no active or viable microbe in the matched metagenomic and metatranscriptomic sequencing of CSF specimens after subtracting those also found in normal saline, DNA extraction buffer, and skin swab specimens. In conclusion, our results showed no strong evidence of a colonized microbial community present in the CSF of healthy individuals. IMPORTANCE The microbiome is prevalent throughout human bodies, with profound health implications. However, it remains unclear whether it is present and active in human CSF, which has been long considered aseptic due to the blood-brain barrier. Here, we applied unbiased metagenomic and metatranscriptomic sequencing to detect the presence of a microbiome in CSF collected from 23 pregnant women with matched controls. Analysis of 116 specimens found no strong evidence to support the presence of a colonized microbiome in CSF. Our findings will strengthen our understanding of the internal environment of the CSF in healthy people, which has strong implications for human health, especially for neurological infections and disorders, and will help further disease diagnostics, prevention, and therapeutics in clinical settings.

clinical samples (see DOI: https://doi.org/10.1128/MRA.01446-18). Consider reframing discussion as the finding of this particular species may strengthen the argument of it being a contaminant.
Lines 280-281: please expand on method of random selection (computer generated sequence etc.?).
Lines 263-264: "providing a guide for disease diagnostics" do the authors suggest that a standardised method as used in this paper with sampling of saline and skin flora followed by subtraction is routinely employed in future clinical studies of CSF that use mNGS?
Lines 96-100 would be better placed in the discussion. Lines 211-218 would be better placed in the discussion.
Minor comments Figure 1 text: "replicates" is probably not correct here as I understand that each are individual samples rather than true replicates of the same sample. Figure 2 text: consider expanding "other samples" to provide more complete description of the labels skin, swab, negative etc. as is done in the text for figure 1 so that this figure could stand alone. Figure 3 text: again, consider expanding "negative" to include description (i.e. saline) Although a reasonable label for analysis, "CSF_DNA" is unconventional in body text (e.g. line 121), consider replacing with "CSF-DNA" or "CSF DNA". Similarly some organism names should have "_" removed in body text e.g. "Escherichia_coli" (line 201).
Please carefully review spelling for example line 180 "substracted" -> "subtracted"; line 320 "palet" -> "pellet"; line 360 "equencing" -> "sequencing"; line 379 " organismal relative abundance" -> "relative abundance of oragnisms"; line 528 "inlet" -> "inset"; line 545 "remined" -> "remained" Reviewer #3 (Comments for the Author): Summary "Cerebrospinal fluids from health pregnant women does not harbor a detectable microbial community" is a prospective study that aimed to describe the cerebrospinal fluid microbiome in healthy individuals. The study used CSF samples from 23 pregnant women aged 23-40 that were undergoing spinal anesthesia prior to C-section. Women were excluded from the study if they had other known infection or inflammatory condition, neurologic disease, hypertension, diabetes, heart disease or cancer. CSF was collected at the time of spinal anesthesia. Additionally skin and oral surface swabs were obtained to use as positive controls. A negative control of saline was collected in a syringe at the time of CSF collection. Overall the study addresses an interesting question of whether or not a microbiome exists in the CSF. However, there are some study design issues that are not addressed in the manuscript and the manuscript would benefit greatly from review by an individual with a thorough knowledge of English grammar.
Major comments 1. While the manuscript addresses an important questions the study includes a very low number of study subjects and all of these participants are pregnant, adult, females. This small population significantly impairs the generalizability of the results of the study since only one gender is being examined in a narrow age group. Additionally, there are immunologic changes during pregnancy so that the growing fetus is tolerated by the mother. I fully understand how difficult it is to obtain CSF from normal health controls as this is an invasive procedure, however, the rationale for the convenience sample that was used in this study should be provided. 2. The manuscript would benefit from a more through discussion of the study design, this would help alleviate the above concern and bring overall clarity to the manuscript. It is unclear over what time period the study was conducted and how many women were considered but excluded based on the exclusion criteria. It would be helpful to discuss why the "n" of the study was 23. While it appears all 23 subjects had samples collected, only "twelve CSF samples were randomly selected for metastrascriptome studies (lines 280-281). It is extremely unclear if the final data set includes an "n" of 23 or 12. 3. The manuscript would benefit greatly from review by an individual with a thorough knowledge of English grammar. As it is written now the errors significantly impact the readability of the manuscript. Microbiome is frequently used in the incorrect context, cerebrospinal fluid should be used rather than cerebrospinal fluids, etc. There is inconsistent phrasing throughout the manuscript and the reference to positive, negative and contamination controls is not well delineated. At times CSF_DNA is used and this seems more like an input variable than an appropriate abbreviation for a manuscript. The methods, particularly sections on DNA extractions and purification, Metagenomics library construction and RNA library preparation for metatranscriptomics sequencing use a numbered approach that reads more like the steps in a protocol than a cohesive description of methods. 4. The manuscript would benefit from a more through discussion of the results, some speculation about the viruses and their potential pathogenesis in humans would be beneficial. Since the only significant results in the CSF was that of Aspergillus DNA fragments, this deserves a more in-depth discussion as Aspergillus is a significant human pathogen.
Minor comments 1. The manuscript would also benefit from a more through discussion of the "contamination" controls. These were briefly mentioned but not well outlined in the study design. 2. A more thorough discussion of how the various types of controls were used to exclude many of the organisms found in the CSF and more significant referencing of this method would be helpful. 3. While surface swabs of the skin and mouth were used as a positive control in this study, there was no discussion of if serum/blood would be a more appropriate positive control. Serum/blood would in some ways be much more similar to CSF in terms of composition and it is also technically thought to be sterile. 4. Figure 1 a is very busy, it would be most helpful to have a figure illustrating the various samples and overall study design. Perhaps a flow chart would be more appropriate as a standalone figure rather than inclusion with 1b-d. 5. Overall the size of the text in the figures is difficult to read as it is often very small.

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Summary "Cerebrospinal fluids from health pregnant women does not harbor a detectable microbial community" is a prospective study that aimed to describe the cerebrospinal fluid microbiome in healthy individuals. The study used CSF samples from 23 pregnant women aged 23-40 that were undergoing spinal anesthesia prior to C-section. Women were excluded from the study if they had other known infection or inflammatory condition, neurologic disease, hypertension, diabetes, heart disease or cancer. CSF was collected at the time of spinal anesthesia. Additionally skin and oral surface swabs were obtained to use as positive controls. A negative control of saline was collected in a syringe at the time of CSF collection.
Overall the study addresses an interesting question of whether or not a microbiome exists in the CSF. However, there are some study design issues that are not addressed in the manuscript and the manuscript would benefit greatly from review by an individual with a thorough knowledge of English grammar.
Major comments 1. While the manuscript addresses an important questions the study includes a very low number of study subjects and all of these participants are pregnant, adult, females. This small population significantly impairs the generalizability of the results of the study since only one gender is being examined in a narrow age group. Additionally, there are immunologic changes during pregnancy so that the growing fetus is tolerated by the mother. I fully understand how difficult it is to obtain CSF from normal health controls as this is an invasive procedure, however, the rationale for the convenience sample that was used in this study should be provided. 2. The manuscript would benefit from a more through discussion of the study design, this would help alleviate the above concern and bring overall clarity to the manuscript. It is unclear over what time period the study was conducted and how many women were considered but excluded based on the exclusion criteria. It would be helpful to discuss why the "n" of the study was 23. While it appears all 23 subjects had samples collected, only "twelve CSF samples were randomly selected for metastrascriptome studies (lines 280-281). It is extremely unclear if the final data set includes an "n" of 23 or 12. 3. The manuscript would benefit greatly from review by an individual with a thorough knowledge of English grammar. As it is written now the errors significantly impact the readability of the manuscript. Microbiome is frequently used in the incorrect context, cerebrospinal fluid should be used rather than cerebrospinal fluids, etc. There is inconsistent phrasing throughout the manuscript and the reference to positive, negative and contamination controls is not well delineated. At times CSF_DNA is used and this seems more like an input variable than an appropriate abbreviation for a manuscript. The methods, particularly sections on DNA extractions and purification, Metagenomics library construction and RNA library preparation for metatranscriptomics sequencing use a numbered approach that reads more like the steps in a protocol than a cohesive description of methods. 4. The manuscript would benefit from a more through discussion of the results, some speculation about the viruses and their potential pathogenesis in humans would be beneficial. Since the only significant results in the CSF was that of Aspergillus DNA fragments, this deserves a more indepth discussion as Aspergillus is a significant human pathogen.
1. The manuscript would also benefit from a more through discussion of the "contamination" controls. These were briefly mentioned but not well outlined in the study design. 2. A more thorough discussion of how the various types of controls were used to exclude many of the organisms found in the CSF and more significant referencing of this method would be helpful. 3. While surface swabs of the skin and mouth were used as a positive control in this study, there was no discussion of if serum/blood would be a more appropriate positive control. Serum/blood would in some ways be much more similar to CSF in terms of composition and it is also technically thought to be sterile. 4. Figure 1 a is very busy, it would be most helpful to have a figure illustrating the various samples and overall study design. Perhaps a flow chart would be more appropriate as a standalone figure rather than inclusion with 1b-d.

Overall the size of the text in the figures is difficult to read as it is often very small.
Thank you for all your valuable comments. We have provided a response letter addressing all the issues raised by the reviewers. For clarity, all reviewer comments or quoted contents are in italicized fonts. A point-to-point response to each comment is provided in normal fonts. References to revised manuscript contents are also provided where needed. Please notice that the Figure

RESPONSE:
Thanks for your advice. To clarify the results concisely, we carefully revised the number of microbial taxa mentioned in the results section using nonredundant microbial taxa (lines 104-110, pages 5-6; lines 120-122, page 6; lines 131-133, page 7). To illustrate the difference of numbers caused by microbial types, we added a comparison of the number of redundant and nonredundant microbes in Fig. S2 in the supplemental material, and affirmed their definitions in the methods section (lines 382-385, page 18) and Fig. S2, respectively. All revisions were marked in the manuscript. 2. Although Aspergillus species are common opportunistic pathogens, Aspergillus turcosus has very rarely been reported from clinical samples (see DOI: https://doi.org/10.1128/MRA.01446-18). Consider reframing discussion as the finding of this particular species may strengthen the argument of it being a contaminant.

RESPONSE:
Considering the suggestion, we have reframed the discussion about Aspergillus species (lines 224-230, page 11).

Lines 280-281: please expand on method of random selection (computer generated sequence etc.?).
RESPONSE: Thanks for your suggestion. We have corrected the description of random selection (lines 291-296, page 14). To validate whether the microbiome, if any detected in CSF, was physiologically active, metatranscriptomic sequencing for 12 of the pregnant women CSF specimens were performed. When processing different batches of CSF specimens, we randomly select specimens for both metagenomic and metatranscriptomic sequencing, and the remaining samples just for metagenomic sequencing. A total of 12 CSF specimens were performed using metagenomic and metatranscriptomic sequencing.
4. Lines 263-264: "providing a guide for disease diagnostics" do the authors suggest that a standardised method as used in this paper with sampling of saline and skin flora followed by subtraction is routinely employed in future clinical studies of CSF that use mNGS?

RESPONSE:
We have modified this sentence at Lines 264-267, page 13. We mean that such findings shall strengthen our understanding of the internal environment of CSF in healthy people, which have great implications to human health especially for neurological disorders and infections, and help the further disease diagnostics, prevention, and therapeutics in clinical settings.

RESPONSE:
Considering the suggestion, we have moved these sentences to the discussion section and revised them carefully (lines 261-264, pages 12-13).
6. Lines 211-218 would be better placed in the discussion.

RESPONSE:
Considering the suggestion, we have moved these sentences to the discussion section and reframed discussion as the finding of Aspergillus species (lines 224-230, page 11).

Minor comments
7. Figure 1 text: "replicates" is probably not correct here as I understand that each are individual samples rather than true replicates of the same sample.

RESPONSE:
Thanks for your advice. The number represented a total of 6 extraction buffer specimens were collected, not the replicates. We have corrected this error of Fig. 1 text. 8. Figure 2 text: consider expanding "other samples" to provide more complete description of the labels skin, swab, negative etc. as is done in the text for figure 1 so that this figure could stand alone.

RESPONSE:
According to the suggestion, we reorganized the original Fig. 2 and made the original Fig. 2b independent as Fig. 4. We have added the description of "other samples" of Fig. 4 text in revised manuscript. Figure 3 text: again, consider expanding "negative" to include description (i.e. saline).

RESPONSE:
Thanks for your advice. We have added the description of "negative" of new Fig. 5 text in revised manuscript.

RESPONSE:
Thanks for your advice. We have deleted the "_" of these words in the revised manuscript.

RESPONSE:
We are very sorry for these spelling errors and have corrected them carefully. The manuscript has been revised by a native English speaker.

Response to reviewer #3 comments:
Major comments 1. While the manuscript addresses an important questions the study includes a very low number of study subjects and all of these participants are pregnant, adult, females. This small population significantly impairs the generalizability of the results of the study since only one gender is being examined in a narrow age group. Additionally, there are immunologic changes during pregnancy so that the growing fetus is tolerated by the mother. I fully understand how difficult it is to obtain CSF from normal health controls as this is an invasive procedure, however, the rationale for the convenience sample that was used in this study should be provided.

RESPONSE:
We are sorry for the limitations of the collected samples in this study. CSF is difficult to obtain in healthy individuals. Only 23 pregnant females who underwent intraspinal anesthesia before the caesarean section were enrolled. Because CSF puncture is an invasive surgery and will cause pain and damage to the subjects. Usually, only patients with CNS infections will receive lumbar punctures. Lumbar puncture in healthy people is rare. We have added this description in our manuscript at lines 254-258, page 12.
2. The manuscript would benefit from a more through discussion of the study design, this would help alleviate the above concern and bring overall clarity to the manuscript. It is unclear over what time period the study was conducted and how many women were considered but excluded based on the exclusion criteria. It would be helpful to discuss why the "n" of the study was 23. While it appears all 23 subjects had samples collected, only "twelve CSF samples were randomly selected for metastrascriptome studies (lines 280-281). It is extremely unclear if the final data set includes an "n" of 23 or 12.

RESPONSE:
Thanks for your advice. A more detailed description of the enrolled individuals, including the time period of sample collection, and the criteria were added in the Material and method section (lines 269-303, pages 13-14). According to the exclusion criteria, we only collected eligible pregnant women for this study, and the number of pregnant women excluded was not counted. We added a flow-chart in the supplemental material (Fig. S1) to illustrate the study design and sample composition. To validate whether the microbiome, if any detected in CSF, was