Variations in the oral microbiome and metabolome of methamphetamine users

ABSTRACT Drug addiction can seriously damage human physical and mental health, while detoxification is a long and difficult process. Although studies have reported changes in the oral microbiome of methamphetamine (METH) users, the role that the microbiome plays in the process of drug addiction is still unknown. This study aims to explore the function of the microbiome based on analysis of the variations in the oral microbiome and metabolome of METH users. We performed the 16S rRNA sequencing analysis based on the oral saliva samples collected from 278 METH users and 105 healthy controls (CTL). In addition, the untargeted metabolomic profiling was conducted based on 220 samples. Compared to the CTL group, alpha diversity was reduced in the group of METH users and the relative abundances of Peptostreptococcus and Gemella were significantly increased, while the relative abundances of Campylobacter and Aggregatibacter were significantly decreased. Variations were also detected in oral metabolic pathways, including enhanced tryptophan metabolism, lysine biosynthesis, purine metabolism, and steroid biosynthesis. Conversely, the metabolic pathways of porphyrin metabolism, glutathione metabolism, and pentose phosphate were significantly reduced. It was speculated that four key microbial taxa, i.e., Peptostreptococcus, Gemella, Campylobacter, and Aggregatibacter, could be involved in the toxicity and addiction mechanisms of METH by affecting the above metabolic pathways. It was found that with the increase of drug use years, the content of tryptamine associated with neuropsychiatric disorders was gradually increased. Our study provides novel insights into exploring the toxic damage and addiction mechanisms underlying the METH addiction. IMPORTANCE It was found that with the increase of drug use years, the content of tryptamine associated with neuropsychiatric disorders gradually increased. The prediction models based on oral microbiome and metabolome could effectively predict the methamphetamine (METH) smoking. Our study provides novel insights into the exploration of the molecular mechanisms regulating the toxic damage and addiction of METH as well as new ideas for early prevention and treatment strategies of METH addiction.

• Upload point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER • Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file • Upload a clean .DOC/.DOCX version of the revised manuscript and remove the previous version • Each figure must be uploaded as a separate, editable, high-resolution file (TIFF or EPS preferred), and any multipanel figures must be assembled into one file • Any supplemental material intended for posting by ASM should be uploaded separate from the main manuscript; you can combine all supplemental material into one file (preferred) or split it into a maximum of 10 files, with all associated legends included For complete guidelines on revision requirements, see our Submission and Review Process webpage.Submission of a paper that does not conform to guidelines may delay acceptance of your manuscript.
Data availability: ASM policy requires that data be available to the public upon online posting of the article, so please verify all links to sequence records, if present, and make sure that each number retrieves the full record of the data.If a new accession number is not linked or a link is broken, provide mSystems production staff with the correct URL for the record.If the accession numbers for new data are not publicly accessible before the expected online posting of the article, publication may be delayed; please contact production staff (mSystems@asmusa.org)immediately with the expected release date.
Publication Fees: For information on publication fees and which article types are subject to charges, visit our website.If your manuscript is accepted for publication and any fees apply, you will be contacted separately about payment during the production process; please follow the instructions in that e-mail.Arrangements for payment must be made before your article is published.
ASM Membership: Corresponding authors may join or renew ASM membership to obtain discounts on publication fees.Need to upgrade your membership level?Please contact Customer Service at Service@asmusa.org.
The ASM Journals program strives for constant improvement in our submission and publication process.Please tell us how we can improve your experience by taking this quick Author Survey.
Thank you for submitting your paper to mSystems.

Sincerely, Christopher Marshall Editor mSystems
Reviewer #1 (Comments for the Author): The authors have investigated the functions (e.g., detoxification) of oral microbiome of methamphetamine (METH) users using various well-established methodologies, including 16S rRNA sequencing and metabolomic profiling.The main discoveries included that the alpha diversity was reduced in the METH users, showing significant differences in the microbiota and changes in oral metabolic pathways.The authors identified four key microbial taxa, i.e., Peptostreptococcus, Gemella, Campylobacter, and Aggregatibacter, involved in the toxicity and addiction mechanisms of METH by affecting the above metabolic pathways.Also, the content of tryptamine associated with neuropsychiatric disorders was gradually increased.
I have carefully reviewed the manuscript and found no major technical concerns.I believe that the authors have provided sufficient background, explained well the methodologies used in this study, presented the data with appropriate figures, and more importantly, withdrawn conclusions based on available data.I have some minor suggestions and a few typos listed here for the authors to consider if a revision is requested by the editor.Title: I would suggest the replacement of "Alterations" with "Variations" Abstract: Line 32: it is not clear what the "in this process" means, detoxification?
Line 40: the authors should indicate either "increase" or "decrease" of "with significant differences" Line 47, delete "And," Line 48: "was gradually increased" Introduction: Line 86: I would suggest that the authors explicitly describe the goals of aims of this study somewhere in this paragraph.
Materials and Methods: Line 105: delete "the"

Results:
The figures are well done, nice and informative.
Discussion: I would suggest that the authors establish two subsections, microbiome and metabolome, to focus on the in-dept discussion of each of these two areas.

Reviewer #2 (Comments for the Author):
This study focused on the oral microbiology and metabonomics in methamphetamine abusers，provideing novel insights into exploring the toxic damage and addiction mechanisms underlying the METH addiction.Identification of fundamental variations in the microbiome and how the host will/can respond to this changing microbial burden is an important area for knowledge acquisition.Generally, this report is well organized and informative.The methodology of the biology is sound and the statistical analysis and bioinformatics visualization of the data is good.However, I do have a number of minor to moderate concerns and one larger issue that adversely affects my enthusiasm for the report.
(1)The oral microbiota is influenced by many factors, such as the whole body and the local part of the mouth.The lifestyle of methamphetamine addicts is more complicated.Will there be differences in oral microbiota caused by oral health or oral hygiene problems?How to eliminate interference?(2)The research object is the patients in the compulsory detoxification center, and the author's topic is "Alterations in the oral microbiome and metabolome of methamphetamine users ", so are the oral microorganisms the same during detoxification and non-detoxification?Has the author considered this problem?(3)Line187 "In the discovery phase (194 METH users and 74 CTL)"，Line189"in the validation phase ( METH 189 users and 31 CTL)"，Line190"In the discovery phase of metabolomics (119 METH users and 35 CTL)"，Line194"based on a total of 51 METH users and 15 CTL"，I would suggest including power calculation description to the above data in the manuscript.
This study focused on the oral microbiology and metabonomics in methamphetamine abusers， provideing novel insights into exploring the toxic damage and addiction mechanisms underlying the METH addiction.Identification of fundamental variations in the microbiome and how the host will/can respond to this changing microbial burden is an important area for knowledge acquisition.Generally, this report is well organized and informative.The methodology of the biology is sound and the statistical analysis and bioinformatics visualization of the data is good.However, I do have a number of minor to moderate concerns and one larger issue that adversely affects my enthusiasm for the report.
(1)The oral microbiota is influenced by many factors, such as the whole body and the local part of the mouth.The lifestyle of methamphetamine addicts is more complicated.Will there be differences in oral microbiota caused by oral health or oral hygiene problems?How to eliminate interference?(2)The research object is the patients in the compulsory detoxification center, and the author's topic is "Alterations in the oral microbiome and metabolome of methamphetamine users ", so are the oral microorganisms the same during detoxification and non-detoxification?Has the author considered this problem?(3)Line187 "In the discovery phase (194 METH users and 74 CTL)"，Line189"in the validation phase ( METH 189 users and 31 CTL)"，Line190"In the discovery phase of metabolomics (119 METH users and 35 CTL)"，Line194 "based on a total of 51 METH users and 15 CTL"，I would suggest including power calculation description to the above data in the manuscript.