Review for 10.1186/1471-230X-9-95 (2009)

Reviewer's report
Title:Percent G+C profiling and sequence analysis reveals significant
differences in the gastrointestinal microbiota between diarrhoea-predominant
irritable bowel syndrome sufferers and healthy individuals
Version: 2Date:24 August 2009
Reviewer: Petra Louis
Reviewer's report:
Krogius-Kurikka et al describe differences in the gut microbiota of health
volunteers and subjects with irritable bowel syndrome by clone library analysis
and real-time PCR. The process of fractionation of DNA before cloning is a
valuable approach in tackling the problem of under-representation of
Actinobacteria in clone libraries. However, I have serious doubts about the
excessive pooling of samples as detailed below.
- Major Compulsory Revisions
1. The comparison of pooled samples representing different numbers of subjects
in the two study groups is likely to lead to skewed results, as the diversity would
be expected to be higher in the sample containing DNA from more individuals
due to inter-individual variation. The authors state in the discussion (p. 17) that
removal of singletons lead to a similar ratio of OTUs between both samples,
however, more abundant species may also differ between subjects. The rank
abundance and Simpson index are in line with a higher diversity in HC for rarer
OTUs (but not only singletons). It is currently being debated whether there is a
core microbiota present in the vast majority of individuals or whether different
species with similar physiological traits can substitute each other in different
individuals. Unless this is conclusively resolved in favour of the 1. hypothesis,
comparing datasets with vastly different numbers of subjects is questionable.
2. Despite the fact that different GC-fractions of faecal DNA varied in their
relative amount of DNA, they were treated equal for clone library analysis (i.e.
different fractions contributed similar numbers of clones). This skews the data
(for relative coverage of diversity in different fractions and abundance) and any
data expressed as a percentage of the total are not very meaningful in my view.
A relative comparison between IBS-D and HC should still be possible if the
relative abundance of the DNA in the various fractions was very similar, but as
this was not the case and due to the difference in number of volunteers (see
comment 1), I doubt that the data presented here are conclusive. Another factor
to consider in this context is whether the presence of human DNA, which may
differ between the two study groups and may dominate in certain GC fractions, is
likely to skew the data as well. As PCR products have been pooled for several
IBS-D fractions before cloning, it is impossible to analyse them separately and
apply correction factors to account for the relative DNA abundance in each
fraction.
3. p. 8, sample processing and DNA isolation. Sample processing can have an
effect on the quality of the DNA obtained from faecal samples (for example, see
Molbak et al 2006 Curr Issues Intestinal Microbiol 7: 29). Especially in view of the
fact that the levels of Bacteroides spp. are very low in the current study, can the
authors exclude an effect of their procedure?
4. p. 12, real-time PCR: please provide additional information: Have the primers
been published previously? If yes, please provide reference. If not, how were
they designed and validated? The amount of faecal DNA used seems to be quite
high. Has it been established that it did not inhibit the amplification?
5. p. 14, diversity indices: While I agree that the Shannon index was slightly
lower for IBS-D at all OTU cut-off levels, the Simpson index looks higly similar to
me for OTU cut-offs of up to about 92%. Last sentence of this paragraph: Is there
a mix-up of Shannon and Simpson (the Shannon index was stable while the
Simpson index rose sharply)? Please also correct in the discussion p. 17-18.
6. p. 15-16: I do not understand the last sentence (p. 15). Clostridium XIV and
Bifidobacteriales don’t belong to the Bacilli and Proteobacteria, respectively? I
assume what is meant is that more unique sequences were found in IBS-D within
Bacilli and Proteobacteria and more unique OTUs in cluster XIV and
Bifidobacteriales?
7. p. 16, real-time PCR: What was the rationale for testing these two groups of
bacteria? According to add. file 4, other bacterial genera and groups were also
significantly higher in IBS-D, e.g. Megasphaera; and bacteria significantly
decreased in IBS and quantitative analysis of wider groups (e.g.
Lachnospiraceae) would also be of interest?
8. p. 19, first paragraph, Ruminococci. I am not clear about the evidence
presented here. How did R. torques-related sequences behave in this study (line
4: ‘this may be seen in the IBS-D sequence data composition’)? Last sentence:
Reference 6 is unpublished? What did reference 21 show? In the context of
mucin-degrading bacteria, was Akkermansia muciniphila found?
9. Table 1 and Fig. 4: The numbers of OTUs and singletons don’t add up? Table
1: IBS-D 302 OTUs, 131 singletons; HC 428 OTUs, 170 singletons. Fig. 4: IBS-D
138+176=314 OTUs; HC 176+264=428 OTUs; The singleton numbers for each
sample are higher in the table than in the figure, which indicates that there were
no shared singletons?
10. Discussion, real-time PCR: Could the normalisation against the amount of
faeces have skewed the results, as the consistency and water content of faecal
samples from diarrhea-predominant IBS sufferers might be different from healthy
subjects? A normalisation against the total bacteria (by using universal primers)
would avoid this.
11. Discussion, real-time PCR: Several previous studies seem to report on
real-time PCR results from the same set of volunteers, which makes it difficult to
obtain a proper overview of how real-time quantitation fits to the clone library
data for this study group. Therefore a more comprehensive discussion on how
the two methods agree/disagree should be given, including previous published
data (at phylum level and finer scale).
- Minor Essential Revisions
1. p. 3, results 1. line: Delete ‘between’ (Significant differences were found..). 3.
line: replace ‘Bacteroides than observed in the control’ with ‘Bacteroides
compared to control’.
2. p. 7, line 5: replace ‘controls’ with ‘control’
3. p. 8, number of PCR cycles: please specify.
4. p. 18, last line: Please delete ‘in’
5. RDB: I assume RDP II is meant? Please change or provide the full length term
for the abbreviation.
6. Please replace ssp by spp throughout.
7. Please provide colour code for add. File 3
8. Please provide full information for reference 6 and 46.
9. Please provide references for the Shannon and Simpson index in Material and
Methods and for the statement about their interpretation in discussion, p. 18.
10. Table 1: please indicate OTU cut-off level of 98% for singletons etc. Please
also add this information to the legend of Fig. 3, 4 and 6.
- Discretionary Revisions
These are recommendations for improvement which the author can choose to
ignore. For example clarifications, data that would be useful but not essential.
1. p. 7, line 17: What type of medication was taken by study participants? Line
22: What did the GI examination entail; could this have affected the microbiota?.