Effects of Smoking and Smoking Cessation on the Intestinal Microbiota

Round 1

Reviewer 1 Report

The manuscript entitled "Effects of Smoking and Smoking Cessation on the Intestinal Microbiota " by Stein et al., is well presented.

However, line number is not assigned and it is difficult to comment on the particular line.

This research article maybe improved further and resubmitted for publication

Following are my suggestion:

1) please give line number while you send for review

2) Cardiovascular disease may be added in the Key words for the ease of the readers

3) IInd para: 1st sentence: please rewrite the sentence

4) Why the samples were stored upto 8 hrs and 5 mins at refrigerator after sample collection 

5) DNA extraction: Did u use direct fecal sample or dilution, please write clearly.

6) Table 1: : why only male data is provided ?

7) please discuss about cardiovascular diseases aspects and with other Supporting data available from GCMS analysis

Followings are my question:

1) why metagenomic data at genera level is not presented which could be useful  to understand the genus or even species of Bacteroides and Firmicutes as we know that microorganisms are genus/species/ strain specific and maybe this impacts the discussion and conclusion in a constructive manner ?

Rest is fine and may be published after improving the above mentioned comments.

Author Response

We thank reviewer 1 for his/her favorable review and helpful suggestions.  His/her requests are addressed below.

1) please give line number while you send for review

Responses are keyed to line and page number; formatting has been done per publisher

2) Cardiovascular disease may be added in the Key words for the ease of the readers

Done.

3) IInd para: 1st sentence: please rewrite the sentence

This has been rewritten and changed to active tense (page 2, lines 10-11)

4) Why the samples were stored upto 8 hrs and 5 mins at refrigerator after sample collection 

Samples were not immediately frozen to avoid freeze-thawing cycles. They were initially collected in the research clinic and transported to the lab as soon as possible (up to 8h) to be aliquoted and properly preserved for DNA extraction and 16S analysis.  This study (Choo et al,Sci Rep. 2015 Nov 17;5:16350;  doi: 10.1038/srep16350; PMID: 26572876) shows that refrigeration (up to 72h) does not result in significant alterations in fecal microbiota diversity or composition.

5) DNA extraction: Did u use direct fecal sample or dilution, please write clearly.

We used direct, undiluted fecal samples.  We clarified this on line 3, line 17 by adding this information.

6) Table 1: : why only male data is provided ?

We have added the percent females to the Table (page 4, line 43)

7) please discuss about cardiovascular diseases aspects and with other Supporting data available from GCMS analysis

The details of the gas chromatography technique re in reference 23.  It was used to measure TMAO.  We added (page 5, lines 41-42 to page 6, line 1) that “TMAO levels did not have significant correlations with any of the cardiovascular disease measures (carotid plaque score, PWV, brachial FMD; data not shown).  ON page 6, lines 12013, we state that “Significant between group differences in changes for the 12 week visit were not observed for cardiovascular disease risk factors, hsCRP, TMAO, PWV, and brachial artery FMD (data not shown).”

Followings are my question:

• why metagenomic data at genera level is not presented which could be useful  to understand the genus or even species of Bacteroides and Firmicutes as we know that microorganisms are genus/species/ strain specific and maybe this impacts the discussion and conclusion in a constructive manner ?

This is a very good point.  In response, we have added genera level data to better explain the Firmicutes findings.  We found no associations with bacteroides genera to explain our observations.  On page 6, lines 1-3 we added significant correlations of exhaled CO with Firmicutes species at baseline: “Exhaled CO levels were associated with the Firmicutes genuses Dorea (rho=0.43 p=0.009) and Streptococcus (rho= -0.47, p=0.022).”  We also added (page 6, lines 18-20) that “Among the Firmicutes, we identified a small but statistically significant decrease in the genera Ruminococcus (pANCOVA=0.027) and a small increase in an undefined genus within the order Closteridiales (p=0.043).  No other significant changes between groups were identified for relative abundances of bacteria at the phylum level.”

Reviewer 2 Report

The paper is well written and the topic is interesting.
The effect of smoking on the gut microbiota is known and these results leave some doubts.
The explanation for this could be the small sample, probably a higher sample could lead to different results. However this limitation is also expressed by the authors.
Could the authors better characterize the participants?
Did they have any pathologies? Which? Were they taking drugs, which ones? Please, may the authors specify? 

Author Response

We thank reviewer 2 for his/her favorable review and helpful suggestions. 

This reviewer asked “Did they have any pathologies? Which? Were they taking drugs, which ones? Please, may the authors specify?”

Participants were current adult smokers.  As on page 2,lines 38-44 “Main exclusion criteria were: stage 5 or greater chronic kidney disease, hospitalization for a stroke, heart attack, congestive heart failure or uncontrolled diabetes mellitus within the past year, and alcohol/substance dependence. This sub-study also excluded individuals with factors known to affect the gut microbiota including: use of systemic antibiotics, corticosteroids, immunomodulators, or commercial probiotics in the last 6 months, major gastrointestinal surgery in the past 5 years, and active inflammatory bowel disease or other gastrointestinal disorders.”

Detailed inclusion-exclusion criteria are below, for the reviewer’s reference:

Inclusion Criteria:

1. live or work 10 miles or less from University Campus
2. plan to stay in the area for the next 12 months
3. ability to read and write in English
4. smoke ≥10 cigarettes per day
5. be ≥18 years old
6. desire to quit smoking, but not be engaged currently in cessation treatment
7. report no use of pipe tobacco, cigars, snuff, e-cigarettes or chew in the last 30 days
8. have reliable phone access
9. if female, not be pregnant and be using an acceptable birth control method.

Exclusion Criteria:

1. current treatment for schizophrenia or a psychotic disorder
2. suicidal ideation in the past 12 months;
3. history of suicidal attempts within the last 10 years
4. on dialysis or stage 5 or greater chronic kidney disease
5. hospitalization for a stroke, heart attack, congestive heart failure or uncontrolled diabetes mellitus within the past year
6. history of alcohol/substance dependence since joining the cohort. 
7. have factors known to affect the gut microbiome
   1. Use of systemic antibiotics (intravenous, intramuscular, or oral)
   2. Use of oral, intravenous, intramuscular, nasal or inhaled corticosteroids
   3. Use of immune stimulating medications (e., methotrexate or immunosuppressive cytotoxic agents)
   4. Use of large doses of commercial probiotics (greater than or equal to 108 cfu or organisms per day). This includes tablets, capsules, lozenges, chewing gum or powders in which probiotic is a primary component (ordinary dietary components such as fermented beverages/milks, yogurts, foods do not apply)
   5. Unstable dietary history during the previous month, which is defined as major changes in diet by eliminating or significantly increasing a major food group
   6. Major surgery of the GI tract in the past five years (not including cholecystectomy or appendectomy) or major bowel resection at any time
   7. Active uncontrolled gastrointestinal disorders or diseases including inflammatory bowel disease such as ulcerative colitis, Crohn's disease, moderate or severe irritable bowel syndrome, persistent, infectious gastroenteritis, colitis or gastritis, persistent or chronic diarrhea of unknown etiology, Clostridium difficile infection (recurrent), untreated Helicobacter pylori infection, or chronic severe constipation.