Review for 10.1186/s12876-022-02301-1 (2022)

Reviewer’s report
Title: 
Effects of bowel preparation on intestinal bacterial associated urine and
faecal metabolites and the associated faecal microbiome.
Version: 
0 
Date: 
11 Jan 2022
Reviewer: 
Alberto Caminero
Reviewer's report:
The manuscript BMGE-D-21-00477 by Powles et al. focused on the impact of
bowel cleansing on the microbiome composition and function. Authors recruited
11 participants and collected feces and urine at three different time lines
(baseline, day 3 and week 6 after treatment). Microbiome (16S rRNA gene
sequencing) and metabolomic (1H NMR spectroscopy) analysis were performed
in these samples. Authors found a reduction in alpha diversity (Shannon index) 3
days following bowel cleansing. No difference between samples at baseline and
6 weeks post cleansing were observed. Analysis of urinary and fecal metabolites
showed no significant impact following bowel cleansing. This is an interesting
and well written study of relevance in the field. Although microbiome analysis
has been studied before and after bowel cleansing, main results are supported by
previous studies and the study included metabolomic analysis in urine for the
first time. However, applicant must address major comments before final
publication. 
- The study presents important limitations that must be acknowledged in the
manuscript. Fecal microbiome analysis has been studied already before and after
bowel cleansing. Although this may reduce the novelty of the study, the
literature is well integrated and discussed in the manuscript. Another limitation is
the number of patients/controls recruited. The study only included 11 samples in
this study. Although authors claim that other studies use a similar number of
samples, the sample size is not ideal for microbial composition and function
analysis. In addition, samples are missed for some patients.
- Procedures and Sample collection must be expanded in the Method section.
Authors must define the "low residue diet" and include the methods/treatment for
bowel cleansing/preparation.
- Eleven subjects were recruited from gastroenterology clinics at St. Mary's
Hospital in London who were due to undergo a colonoscopy. There is not too
much information here regarding these subjects. I noticed that 2 participants
were diagnosed with ulcerative colitis and type 2 diabetes when looking in the
Supplementary Figure 1. This information must be also included in the Methods
section. No information about the other participants is included. Are they healthy
or controls? Why did they undergo for colonoscopy? Were these subjects
diagnosed with other major organic or intestinal conditions (IBS, celiac, etc)?
The info must be included in the manuscript. Two subjects did not provide fecal
samples at the last time point, and one of these subjects also did not provide a
urine sample at the last time point. Please expand the Supplementary Table 1 to
show those subjects missing samples.
- The microbial analysis section in Methods must be expanded. Relevant
information such as PCR conditions or amplified fragment (V3, V4, etc) is
missing. Other important information missing number of reads, how the data was
filtered (or if it wasn't) and if it was cleaned. I encourage the authors to include
the total number of reads as well as mean or minimum and maximum number of
reads for this study. Please define main acronyms at first use (e.i. RDP,
Ribosomal Database Project). 
- Figure 1 shows Shannon index at baseline, 3 days and week 6 after bowel
preparation. Considering that these samples come from the same patients, I
encourage the authors to plot dots representing each individual fecal sample and
lines linking the dots for each donor in the different sampling dates, as they did
for beta diversity plot in Figure 2. These will provide a better picture of the data
to the reader.
- Figure 4 shows PCA scores plot of urine metabolic profiles of the 11 subjects.
The Figure shows intra-subject clustering in samples from 9 out of 10 subjects
over the 3 time points. However, some donors such as 4, 5, 9 present some
variations in a single fecal sample. I wonder whether these variations may be
associated to the microbial changes observed in day 3. I encourage the authors to
show the sampling time for each dot in Figure 4 and Supplementary Figure 1.
- Metabolomics was performed on 10 fecal samples at baseline and 10 samples 3
days post colonoscopy. Samples collected at day 6 are missing. There is not
information because these samples were not analyzed.
- Targeted analysis was performed on metabolites and there were no significant
differences between time points after correction for multiple comparisons. The
low sample size has obviously impacted this analysis. I noticed that some
metabolites such as alanine in urine or butyrate in feces are significant before
correction. Although authors used a proper analysis, I would mention or
highlight these metabolites in the manuscript. Future studies could benefit of this
observation. 
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Is the study design appropriate to answer the research question (including the use
of appropriate controls), and are the conclusions supported by the evidence
presented?
Yes
Are the methods sufficiently described to allow the study to be repeated?
No
Is the use of statistics and treatment of uncertainties appropriate?
Yes
Is the presentation of the work clear?
Yes
Are the images in this manuscript (including electrophoretic gels and blots) free
from apparent manipulation?
Yes