Reviewer's report
Title:Lactobacillus acidophilus CRL 1014 improved gut health in the SHIME
reactor
Version: 2Date:24 November 2012
Reviewer: Susana Fuentes
Reviewer's report:
Sivieri and co-authors report on changes in microbial composition and metabolic
activity in an in vitro model of the human gastrointestinal tract, the SHIME model,
in response to treatment with a probiotic strain of Lactobacillus acidophilus. In
general, the manuscript describes an interesting study, as it addresses the effect
of probiotic bacteria on overall microbial community composition and function.
I do, however, have a number of issues with the manuscript as pointed out in
more detail in the following.
1) I have some reservations regarding the type and presentation of microbial
composition analysis. First of all, the authors mention total bacterial community
analysis by DGGE profiling of PCR-amplified 16S rRNA gene fragments,
however, this is not shown. At some point they refer to Fig. 2, however, that
figure only shows Lactobacillus specific profiles.
Secondly, the authors claim, e.g. in the abstract, that their data show that the
administered strain successfully colonized the lower intestine. I have to disagree
with the authors here, as they don’t provide any direct evidence for that. Plate
counts are done for lactobacilli in general. Furthermore, L.acidophilus was not
represented by any of the sequenced DGGE bands. To this end, I would suggest
to repeat the DGGE analysis, now including a PCR product generated from DNA
of the probiotic strain as a positive control.
Also, regarding the plate counts, I would like to see data split out for the different
sampling points in order to see whether numbers are stable during the three
periods. Especially, I would like to see evidence for the stability of microbiota
prior to the addition of the probiotic.
2) The authors did not perform any unoculated control experiment. I would like to
see evidence (e.g. from previous experiments performed with this model under
comparable conditions) that nothing changes over an experimental period of 8
weeks when no specific treatment is included.
Specific comments:
First of all, I would like to indicate that it would have been helpful if the authors
had included page and line numbers. Please make this a default habit in order to
facilitate the communication between reviewers and authors. I have now
numbered pages starting with the title page, and the abstract on page 2.
p.2, l.10: Be more specific regarding the probiotic administration (i.e. daily dose)
p.2: be more specific regarding the locations where differences in metabolite
concentrations were found
p.3, l.16: Lactobacillus spp. are really not so numerous in the lower intestinal
tract.
p.3, l.18: Enterobacteriaceae should be in italics. Same is true for Lactobacillus
acidophilus elsewhere throughout the manuscript.
p.4, l.12: Rephrase: Cultures were harvested during the exponential growth
phase (OD??)
Fig. 1: Make this a supplementary figure, and add Tables 1 and 2 to the main
manuscript instead.
p.5, from l.6: Seems that this paragraph contains the same info twice (first two
sentences).
p.5, l.25: is this really specifically Enterobacterium? I would think that it’s
generally enterobacteria, including e.g. E.coli?
p.5: reference 20 is missing in the text
p.7: References Lane (1991), Muyzer (1993) and Sanguinetti (1994) have to be
added to the reference list
p.8, l.26: The authors write that they didn’t observe any effects on lactobacilli and
bifidobacteria during the blank period. Effects compared to what?
p.9, l.5: The authors say that there is no significant changes, but then p<0.01?
p.9, in the middle: A similarity of 70% is not so low actually! In general, I have the
impression that the authors overinterprete small differences in similarities.
Fig.2/3: I don’t see any reason why the authors decided to split these two figures.
p.9, last line: 81% is very low. Is this due to bad sequence quality?
p.10, l.10: These measurements were used to characterize microbial
metabolism, rather than microbial populations
p.11: middle: Is the cultivation used here specific for Clostridium spp., or more
general for members of the different Clostridium clusters, which also include
many other genera?
p.11, l.24: How do we know that the effect is positive?
Reference 5 is missing in the list
Fig. 4: Legend in the figure is in Portuguese
Fig. 5: Include lactic acid in the figure legend
Level of interest: An article whose findings are important to those with closely
related research interests
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:
no competing interests
Reviewer's report
Title:Lactobacillus acidophilus CRL 1014 improved gut health in the SHIME
reactor
Version: 2Date:24 November 2012
Reviewer: Susana Fuentes
Reviewer's report:
Sivieri and co-authors report on changes in microbial composition and metabolic
activity in an in vitro model of the human gastrointestinal tract, the SHIME model,
in response to treatment with a probiotic strain of Lactobacillus acidophilus. In
general, the manuscript describes an interesting study, as it addresses the effect
of probiotic bacteria on overall microbial community composition and function.
I do, however, have a number of issues with the manuscript as pointed out in
more detail in the following.
1) I have some reservations regarding the type and presentation of microbial
composition analysis. First of all, the authors mention total bacterial community
analysis by DGGE profiling of PCR-amplified 16S rRNA gene fragments,
however, this is not shown. At some point they refer to Fig. 2, however, that
figure only shows Lactobacillus specific profiles.
Secondly, the authors claim, e.g. in the abstract, that their data show that the
administered strain successfully colonized the lower intestine. I have to disagree
with the authors here, as they don’t provide any direct evidence for that. Plate
counts are done for lactobacilli in general. Furthermore, L.acidophilus was not
represented by any of the sequenced DGGE bands. To this end, I would suggest
to repeat the DGGE analysis, now including a PCR product generated from DNA
of the probiotic strain as a positive control.
Also, regarding the plate counts, I would like to see data split out for the different
sampling points in order to see whether numbers are stable during the three
periods. Especially, I would like to see evidence for the stability of microbiota
prior to the addition of the probiotic.
2) The authors did not perform any unoculated control experiment. I would like to
see evidence (e.g. from previous experiments performed with this model under
comparable conditions) that nothing changes over an experimental period of 8
weeks when no specific treatment is included.
Specific comments:
First of all, I would like to indicate that it would have been helpful if the authors
had included page and line numbers. Please make this a default habit in order to
facilitate the communication between reviewers and authors. I have now
numbered pages starting with the title page, and the abstract on page 2.
p.2, l.10: Be more specific regarding the probiotic administration (i.e. daily dose)
p.2: be more specific regarding the locations where differences in metabolite
concentrations were found
p.3, l.16: Lactobacillus spp. are really not so numerous in the lower intestinal
tract.
p.3, l.18: Enterobacteriaceae should be in italics. Same is true for Lactobacillus
acidophilus elsewhere throughout the manuscript.
p.4, l.12: Rephrase: Cultures were harvested during the exponential growth
phase (OD??)
Fig. 1: Make this a supplementary figure, and add Tables 1 and 2 to the main
manuscript instead.
p.5, from l.6: Seems that this paragraph contains the same info twice (first two
sentences).
p.5, l.25: is this really specifically Enterobacterium? I would think that it’s
generally enterobacteria, including e.g. E.coli?
p.5: reference 20 is missing in the text
p.7: References Lane (1991), Muyzer (1993) and Sanguinetti (1994) have to be
added to the reference list
p.8, l.26: The authors write that they didn’t observe any effects on lactobacilli and
bifidobacteria during the blank period. Effects compared to what?
p.9, l.5: The authors say that there is no significant changes, but then p<0.01?
p.9, in the middle: A similarity of 70% is not so low actually! In general, I have the
impression that the authors overinterprete small differences in similarities.
Fig.2/3: I don’t see any reason why the authors decided to split these two figures.
p.9, last line: 81% is very low. Is this due to bad sequence quality?
p.10, l.10: These measurements were used to characterize microbial
metabolism, rather than microbial populations
p.11: middle: Is the cultivation used here specific for Clostridium spp., or more
general for members of the different Clostridium clusters, which also include
many other genera?
p.11, l.24: How do we know that the effect is positive?
Reference 5 is missing in the list
Fig. 4: Legend in the figure is in Portuguese
Fig. 5: Include lactic acid in the figure legend
Level of interest: An article whose findings are important to those with closely
related research interests
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:
no competing interests
Please review this paper.
user
Reviewer's report
Title:Lactobacillus acidophilus CRL 1014 improved gut health in the SHIME
reactor
Version: 2Date:24 November 2012
Reviewer: Susana Fuentes
Reviewer's report:
Sivieri and co-authors report on changes in microbial composition and metabolic
activity in an in vitro model of the human gastrointestinal tract, the SHIME model,
in response to treatment with a probiotic strain of Lactobacillus acidophilus. In
general, the manuscript describes an interesting study, as it addresses the effect
of probiotic bacteria on overall microbial community composition and function.
I do, however, have a number of issues with the manuscript as pointed out in
more detail in the following.
1) I have some reservations regarding the type and presentation of microbial
composition analysis. First of all, the authors mention total bacterial community
analysis by DGGE profiling of PCR-amplified 16S rRNA gene fragments,
however, this is not shown. At some point they refer to Fig. 2, however, that
figure only shows Lactobacillus specific profiles.
Secondly, the authors claim, e.g. in the abstract, that their data show that the
administered strain successfully colonized the lower intestine. I have to disagree
with the authors here, as they don’t provide any direct evidence for that. Plate
counts are done for lactobacilli in general. Furthermore, L.acidophilus was not
represented by any of the sequenced DGGE bands. To this end, I would suggest
to repeat the DGGE analysis, now including a PCR product generated from DNA
of the probiotic strain as a positive control.
Also, regarding the plate counts, I would like to see data split out for the different
sampling points in order to see whether numbers are stable during the three
periods. Especially, I would like to see evidence for the stability of microbiota
prior to the addition of the probiotic.
2) The authors did not perform any unoculated control experiment. I would like to
see evidence (e.g. from previous experiments performed with this model under
comparable conditions) that nothing changes over an experimental period of 8
weeks when no specific treatment is included.
Specific comments:
First of all, I would like to indicate that it would have been helpful if the authors
had included page and line numbers. Please make this a default habit in order to
facilitate the communication between reviewers and authors. I have now
numbered pages starting with the title page, and the abstract on page 2.
p.2, l.10: Be more specific regarding the probiotic administration (i.e. daily dose)
p.2: be more specific regarding the locations where differences in metabolite
concentrations were found
p.3, l.16: Lactobacillus spp. are really not so numerous in the lower intestinal
tract.
p.3, l.18: Enterobacteriaceae should be in italics. Same is true for Lactobacillus
acidophilus elsewhere throughout the manuscript.
p.4, l.12: Rephrase: Cultures were harvested during the exponential growth
phase (OD??)
Fig. 1: Make this a supplementary figure, and add Tables 1 and 2 to the main
manuscript instead.
p.5, from l.6: Seems that this paragraph contains the same info twice (first two
sentences).
p.5, l.25: is this really specifically Enterobacterium? I would think that it’s
generally enterobacteria, including e.g. E.coli?
p.5: reference 20 is missing in the text
p.7: References Lane (1991), Muyzer (1993) and Sanguinetti (1994) have to be
added to the reference list
p.8, l.26: The authors write that they didn’t observe any effects on lactobacilli and
bifidobacteria during the blank period. Effects compared to what?
p.9, l.5: The authors say that there is no significant changes, but then p<0.01?
p.9, in the middle: A similarity of 70% is not so low actually! In general, I have the
impression that the authors overinterprete small differences in similarities.
Fig.2/3: I don’t see any reason why the authors decided to split these two figures.
p.9, last line: 81% is very low. Is this due to bad sequence quality?
p.10, l.10: These measurements were used to characterize microbial
metabolism, rather than microbial populations
p.11: middle: Is the cultivation used here specific for Clostridium spp., or more
general for members of the different Clostridium clusters, which also include
many other genera?
p.11, l.24: How do we know that the effect is positive?
Reference 5 is missing in the list
Fig. 4: Legend in the figure is in Portuguese
Fig. 5: Include lactic acid in the figure legend
Level of interest: An article whose findings are important to those with closely
related research interests
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:
no competing interests
assistant