Review for 10.1186/1471-230X-14-40 (2014)

Reviewer's report
Title:Changes in Gut Bacterial Populations and Their Translocation into Liver
and Ascites in Alcoholic Liver Cirrhotics
Version:1Date:28 October 2013
Reviewer: Anne Salonen
Reviewer's report:
Changes in Gut Bacterial Populations and Their Translocation into Liver and
Ascites in Alcoholic Liver Cirrhotics by Tuomisto et al.
Review comments to the manuscript
The paper studies the involvement of intestinal bacteria, and their translocation
from gut to liver in the development of alcoholic liver cirrhosis. This is medically a
highly relevant question, as bacteria may explain why only a subset of alcoholics
develop liver cirrhosis. The authors address three main questions: 1) Does gut
microbiota differ in cirrhotic- and non-cirrhotic alcoholics, and healthy controls,
using fecal matter originating from autopsies and live individuals 2) do liver (in
autopsies) and ascites (in live individuals) contain intestinal bacteria, reflecting
translocation, and 3) is the expression of LPS-binding CD14 affected by the
phenotype in the autopsy livers, providing additional evidence for translocation.
The presence and identity of bacteria were studies by standard blood culturing
and real time PCR, which is more sensitive and detects both live and dead
bacteria in the samples.
Generally the presented data is adequate. However, the validity of the first
approach cannot be evaluated with the available information. Firstly, there are 5
study groups, as presented in Table 1. Yet results are presented for only 4
groups (Abstract, Figure 1 and text). According to abstract, the live cirrhotics are
missing. All statistically significant differences are detected between healthy live
volunteers, and the presented cirrhotic samples. Assuming all the cirrhotics are
autopsy samples, the impact of pathology and post mortem sampling cannot be
differentiated. Although the authors have performed a study on time-dependent
post mortem changes of the microbiota, the results were not available for review
(ref 17, submitted), neither were the amount of total bacteria reported and
compared between feces collected form decades and volunteers.
The manuscript adheres to relevant standards for reporting. No data was
deposited. The discussion and conclusions are adequately supported by the
data. The acknowledgement of previous work is appropriate, however two
missing but relevant references are specified below. The limitations of the work
are not stated. The title and abstract accurately describe the work, yet small
clarifications are required in the terminology of the abstract as specified below.
Writing is fluent English.
Major Compulsory Revisions (which the author must respond to before a
decision on publication can be reached)
Entire manuscript:
1. There are 5 study groups, as presented in Table 1. Yet data is presented for
only 4 groups (Figure 1 and text). Apparently feces of live cirrhotic patients were
not analyzed for bacterial content. Please clarify.
2. In the abstract (first two sentences in the results part) and in the results, the
authors refer to feces and rectum samples so that it is not clear, whether these
refer to same or different type of biological sample. Furthermore, in material and
methods, it is mentioned that rectum and cecum samples were collected from
autopsies. The latter is not mentioned afterwards. Were samples from these two
cites pooled, or the latter not analyzed? Please clarify, taking into account the
results of your submitted comparative study: If the post mortem samples cannot
be reliably compared to feces, refer to these samples with different terminology.
If they are comparable, please explain this and thereafter refer the gut content
consistently to as feces, whether originating from dead or live individual.
3. Not patients, but only healthy individuals are termed volunteers. As both
cirrhotics and healthy controls signed a consent, please refer to both of these
groups as volunteers with specification of health status when relevant.
Background, 2nd paragraph, and Discussion, 1st paragraph:
4. Enterobacteriacea is not a phylum, but a family. Please correct.
Minor Essential Revisions (such as missing labels on figures, or the wrong use of
a term, which the author can be trusted to correct)
Background, 2nd paragraph:
5. Please mention first the Clostridium Clusters XIVa and IV as they are
numerically much more dominant than the 3 bacterial groups that are now
mentioned first.
6. When referring to reference 9, the authors should indicate that alcoholics were
studied; the current phrasing “ingested alcohol has been shown to change
intestinal bacterial population” gives an impression of short-term, acute effect. In
the same sentence, the authors should mention that the amount of Bacteroidetes
(gram-negative, anaerobe) was decreased in alcoholics, as this was the major
result of the paper.
Background, 3rd paragraph:
7. For C. leptum, please add term “group” to indicate that not a single species but
a large bacterial groups was targeted. Please correct this also in Materials and
Methods in paragraph describing PCR.
Materials and methods, PCR paragraph:
8. Should be “all the primers” for specificity and cross reactivity
Materials and methods, statistics:
9. Please spell out the LSD correction.
Results, 1st sentence:
10. Please use term “comparable” instead of “similar” to describe the bacterial
counts in non-cirrhotic autopsies and healthy controls.
Results, 2nd paragraph:
11. Instead of reporting DNA-negativity, a more intuitive way would be to report
the percentage of (translocation-) positive samples.
Results, last paragraph:
12. Please replace “as well as ” with “or” in the second last sentence.
13. In the Figure 5, rather than the amount of total bacterial DNA, its presence
was used to categorize the samples. Please correct in the text. Although the
caption of Figure 5 states to visualize correlation, the current figure shows
differential expression of CD14 between the two groups. To visualize correlation,
the authors should use e.g. x-y plot.
Discussion, 1st paragraph:
14. Please cite in parallel to refs 13 and 14 a recent study by Kakiyama et al.
http://www.ncbi.nlm.nih.gov/pubmed/23333527 , which is one of the most
comprehensive microbiota descriptions in cirrhotic patients. As discussed by
these authors, bile acids in general, and cholestasis (blockage of bile flow from
liver), common in cirrhotic patients, may be one mechanistic explanation for the
altered microbiota in liver diseases. Please mention this in your discussion.
Discussion, 2nd paragraph:
15. In the last sentence, please give a reference for CD 14-mediated
inflammation to indicate that it is a well established phenomena, e.g. in context of
insulin resistance-related inflammation.
Discussion, 1st paragraph:
16. Acetaldehyde: effect, not affect
17. Discretionary Revisions (which are recommendations for improvement but
which the author can choose to ignore)
18. How and from where were the healthy volunteers requited?
Background, second paragraph:
19. Susceptible, rather than vulnerable, is used to refer to subjects with genetic
predisposition.
Materials and methods, 2nd paragraph:
20. Please either report the questionnaire data, or remove reference to it.
Level of interest: An article of importance in its field
Quality of written English: Needs some language corrections before being
published
Statistical review: No, the manuscript does not need to be seen by a statistician.
Declaration of competing interests:
I declare that I have no competing interests