title: Mentholation affects the cigarette microbiota by selecting for bacteria resistant to harsh environmental conditions and selecting against potential bacterial pathogens.
abstract: There is a paucity of data regarding the microbial constituents of tobacco products and their impacts on public health. Moreover, there has been no comparative characterization performed on the bacterial microbiota associated with the addition of menthol, an additive that has been used by tobacco manufacturers for nearly a century. To address this knowledge gap, we conducted bacterial community profiling on tobacco from user- and custom-mentholated/non-mentholated cigarette pairs, as well as a commercially-mentholated product. Total genomic DNA was extracted using a multi-step enzymatic and mechanical lysis protocol followed by PCR amplification of the V3-V4 hypervariable regions of the 16S rRNA gene from five cigarette products (18 cigarettes per product for a total of 90 samples): Camel Crush, user-mentholated Camel Crush, Camel Kings, custom-mentholated Camel Kings, and Newport Menthols. Sequencing was performed on the Illumina MiSeq platform and sequences were processed using the Quantitative Insights Into Microbial Ecology (QIIME) software package.
  
  
  
  In all products, Pseudomonas was the most abundant genera and included Pseudomonas oryzihabitans and Pseudomonas putida, regardless of mentholation status. However, further comparative analysis of the five products revealed significant differences in the bacterial compositions across products. Bacterial community richness was higher among non-mentholated products compared to those that were mentholated, particularly those that were custom-mentholated. In addition, mentholation appeared to be correlated with a reduction in potential human bacterial pathogens and an increase in bacterial species resistant to harsh environmental conditions.
  
  
  
  Taken together, these data provide preliminary evidence that the mentholation of commercially available cigarettes can impact the bacterial community of these products.
authors.affiliation: Maryland Institute for Applied Environmental Health, University of Maryland School of Public Health, College Park, MD, USA.
authors.forename: Jessica
authors.initials: J
authors.lastname: Chopyk
authors.affiliation: Maryland Institute for Applied Environmental Health, University of Maryland School of Public Health, College Park, MD, USA.
authors.forename: Suhana
authors.initials: S
authors.lastname: Chattopadhyay
authors.affiliation: Maryland Institute for Applied Environmental Health, University of Maryland School of Public Health, College Park, MD, USA.
authors.forename: Prachi
authors.initials: P
authors.lastname: Kulkarni
authors.affiliation: Maryland Institute for Applied Environmental Health, University of Maryland School of Public Health, College Park, MD, USA.
authors.forename: Emma
authors.initials: E
authors.lastname: Claye
authors.affiliation: Maryland Institute for Applied Environmental Health, University of Maryland School of Public Health, College Park, MD, USA.
authors.forename: Kelsey R
authors.initials: KR
authors.lastname: Babik
authors.affiliation: Maryland Institute for Applied Environmental Health, University of Maryland School of Public Health, College Park, MD, USA.
authors.forename: Molly C
authors.initials: MC
authors.lastname: Reid
authors.affiliation: Maryland Institute for Applied Environmental Health, University of Maryland School of Public Health, College Park, MD, USA.|School of Medicine, Institute for Genome Sciences and Department of Microbiology and Immunology, University of Maryland, 801 West Baltimore Street, Office #622, Baltimore, MD, 21201, USA.
authors.forename: Eoghan M
authors.initials: EM
authors.lastname: Smyth
authors.affiliation: School of Medicine, Institute for Genome Sciences and Department of Microbiology and Immunology, University of Maryland, 801 West Baltimore Street, Office #622, Baltimore, MD, 21201, USA.
authors.forename: Lauren E
authors.initials: LE
authors.lastname: Hittle
authors.affiliation: Center for Bioinformatics and Computational Biology, University of Maryland, College Park, MD, USA.
authors.forename: Joseph N
authors.initials: JN
authors.lastname: Paulson
authors.affiliation: Department of Epidemiology and Biostatistics, University of Maryland School of Public Health, College Park, MD, USA.
authors.forename: Raul
authors.initials: R
authors.lastname: Cruz-Cano
authors.affiliation: Center for Bioinformatics and Computational Biology, University of Maryland, College Park, MD, USA.
authors.forename: Mihai
authors.initials: M
authors.lastname: Pop
authors.affiliation: Public Health Center for Tobacco Research, Battelle, Columbus, OH, USA.
authors.forename: Stephanie S
authors.initials: SS
authors.lastname: Buehler
authors.affiliation: Department of Behavioral and Community Health, University of Maryland School of Public Health, College Park, MD, USA.
authors.forename: Pamela I
authors.initials: PI
authors.lastname: Clark
authors.affiliation: Maryland Institute for Applied Environmental Health, University of Maryland School of Public Health, College Park, MD, USA.
authors.forename: Amy R
authors.initials: AR
authors.lastname: Sapkota
authors.affiliation: School of Medicine, Institute for Genome Sciences and Department of Microbiology and Immunology, University of Maryland, 801 West Baltimore Street, Office #622, Baltimore, MD, 21201, USA. emongodin@som.umaryland.edu.
authors.forename: Emmanuel F
authors.initials: EF
authors.lastname: Mongodin
mesh_terms: D001741:Black or African American; D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity; D004269:DNA, Bacterial; D006801:Humans; D008610:Menthol / Q000032:analysis*; D064307:Microbiota / Q000235:genetics / Q000502:physiology*; D016133:Polymerase Chain Reaction; D011549:Pseudomonas / Q000235:genetics / Q000302:isolation & purification; D012336:RNA, Ribosomal, 16S; D012907:Smoking; D062789:Tobacco Products / Q000032:analysis / Q000382:microbiology*
chemical_list: D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; D008610:Menthol
keywords: 16S rRNA; Cigarette; Menthol; Microbiota; Pathogens; Tobacco